Method for the inhibition of ALDH-I useful in the treatment of alcohol dependence or alcohol abuse

ABSTRACT

Method for inhibiting aldehyde dehydrogenase activity using daidzin and/or daidzin analog and/or daidzin or daidzin analog in combination with a factor or factors which increase the bioavailability of the daidzin or daidzin analog, as ALDH-I inhibitory compounds or compositions. Such inhibitory compounds or compositions are useful as pharmaceutical compositions in methods for the treatment of alcohol dependence (i.e., alcoholism) or alcohol abuse, for alcohol sensitization, for extinguishing an alcohol-drinking response, for suppressing an urge for alcohol, for inducing alcohol intolerance, for preventing alcoholism in an individual with or without a susceptibility or predisposition to alcoholism or alcohol abuse, and for limiting alcohol consumption in an individual whether or not genetically predisposed.

This application is a § 371 application of PCT/US92/05598 filed Jun. 30,1992 which is a continuation-in-part of Ser. No. 07/723,404, filed Jul.1, 1991, now U.S. Pat. No. 5,204,369.

BACKGROUND OF THE INVENTION

Alcohol abuse and alcohol dependence (i.e., alcoholism) are seriouspublic health problems of modern society. In the United States alone, anestimated 13 million adults exhibit symptoms of alcohol dependence dueto excessive alcohol intake, and an additional 7 million abuse alcoholwithout showing symptoms of dependence according to U.S. Governmentprojections from studies conducted in the mid-1980s. Alcohol dependenceand abuse are very expensive: in economic and medical terms, it willcost the U.S. well over $200 billion in 1991 with no prospect of fallingor leveling off. The social and psychological damages inflicted onindividuals as a consequence of alcohol abuse, e.g., children born withfetal alcohol syndrome (FAS) and victims of alcohol-related accidentaldeath, homicide, suicide, etc., are immense.

While it is generally accepted that alcoholism and alcohol abuse areafflictions with staggering international economic, social, medical, andpsychological repercussions, success in preventing or otherwiseameliorating the consequences of these problems has been an elusivegoal. Only very recently the public view that alcoholism and alcoholabuse are remediable solely by moral imperatives has been changed toinclude an awareness of alcoholism and alcohol abuse as physiologicalaberrations whose etiology may be understood and for which therapy maybe found through scientific pursuits. Both alcohol abuse and dependencearise as a result of different, complex, and as yet incompletelyunderstood processes. At present, alcohol research is in the mainstreamof scientific efforts.

Our studies on alcohol (ethanol or ethyl alcohol) have been based on thehypothesis that its abuse can ultimately be understood and dealt with atthe molecular level. Such a molecular understanding, if achieved, wouldprovide a basis for the identification and development of appropriatetherapeutic agents. Our view hypothesizes that the clinicalmanifestations of alcoholism and alcohol abuse are the consequence ofaberrations or defects within one or more metabolic pathways, affectedby the presence of ethyl alcohol. In order to test this hypothesis, ourinitial studies focused on physical, chemical, and enzymatic propertiesof human alcohol dehydrogenase (ADH), the enzyme that catalyzes alcoholoxidation according to the following reaction formula:

    CH.sub.3 CH.sub.2 OH+NAD.sup.+ →CH.sub.3 CHO+NADH

In addition, our studies more recently have focused on the aldehydedehydrogenases (ALDH) which catalyze the subsequent step in the majorpathway of ethanol metabolism according to the following reactionformula:

    CH.sub.3 CHO+NAD.sup.+ →CH.sub.3 COOH+NADH

Prior to our research (for example, see Blair and Vallee, 1966,Biochemistry 5:2026-2034), ADH in man was thought to exist in but one ortwo forms, primarily in the liver, where it was considered the exclusiveenzyme for the metabolism of ethanol. Currently, four different classesof ADH encompassing over twenty ADH isozymes have been identified andisolated from human tissues. There is no reason to believe that all ofthese ADH isozymes are necessary to catalyze the metabolism of a singlemolecule, ethanol, even though all of them can interact with it. We haveproposed that the normal function of these isozymes is to metabolizeother types of alcohols that participate in critical, physiologicallyimportant processes, and that ethanol interferes with their function(Vallee, 1966, Therapeutic Notes 14:71-74). Further, we predicted thatindividual differences in alcohol tolerance might well be based on bothqualitative and quantitative differences in isozyme endowment (Vallee,1966, supra).

Our research has established the structures, properties, tissuedistribution, and developmental changes for most of the ADH isozymes,which while structurally quite similar, and presumed to have evolvedfrom a common precursor, are functionally remarkably varied. Of the morethan 120 publications from our laboratory that relate to the abovesubjects, the following, arranged in six categories, are especiallyuseful for instruction in the prior art.

(i) Discovery of isozymes: Bosron et al., 1977, "Isolation andCharacterization of an Anodic Form of Human Liver AlcoholDehydrogenase," Biochem. Biophys. Res. Comm. 74:85-91; Bosron et al.,1979, "Human Liver π-Alcohol Dehydrogenase: Kinetic and MolecularProperties," Biochemistry 18:1101-1105; Bosron et al., 1980, "NewMolecular Forms of Human Liver Alcohol Dehydrogenase: Isolation andCharacterization of ADH (Indianapolis)," Proc. Natl. Acad. Sci. USA77:5784-5788; Par es and Vallee, 1981, "New Human Liver AlcoholDehydrogenase Forms with Unique Kinetic Characteristics," Biochem.Biophys. Res. Comm. 98, No. 1:122-130.

(ii) Discovery of new physiological and toxicological substrates: Wackeret al., 1965, "Treatment of Ethylene Glycol Poisoning with EthylAlcohol," JAMA 194:1231-1233; Frey and Vallee, 1980, "DigitalisMetabolism and Human Liver Alcohol Dehydrogenase," Proc. Natl. Acad.Sci. USA 77:924-927; M ardh et al., 1985, "Human Class I AlcoholDehydrogenases Catalyze the Oxidation of Glycols in the Metabolism ofNorepinephrine," Proc. Natl. Acad. Sci. USA 82:4979-4982; M ardh et al1986a, "Testosterone Allosterically Regulates Ethanol Oxidation by Homo-and Heterodimeric γ-Subunit-Containing Isozymes of Human AlcoholDehydrogenase," Proc. Natl. Acad. Sci. USA 83:2836-2840; Consalvi etal., 1986, "Human Alcohol Dehydrogenases and Serotonin Metabolism,"Biochem. Biophys. Res. Comm. 139:1009-1016; M ardh and Vallee, 1986b,"Human Class I Alcohol Dehydrogenases Catalyze the Interconversion ofAlcohols and Aldehydes in the Metabolism of Dopamine," Biochemistry25:7279-7282; M ardh et al., 1986c, "Human Class II (π) AlcoholDehydrogenase Has a Redox-Specific Function in NorepinephrineMetabolism," Proc. Natl. Acad. Sci USA 83:8908-8912; M ardh et al.,1987, "Thyroid Hormones Selectively Modulate Human Alcohol DehydrogenaseIsozyme Catalyzed Ethanol Oxidation," Biochemistry 26:7585-7588; McEvilyet al., 1988, "3β-Hydroxy-5β-steroid Dehydrogenase Activity of HumanLiver Alcohol Dehydrogenase Is Specific to γ-Subunits," Biochemistry27:4284-4288; Keung, 1991, "Human Liver Alcohol Dehydrogenases Catalyzethe Oxidation of the Intermediary Alcohols of the Shunt Pathway ofMevalonate Metabolism," Biochem. Biophys. Res. Comm. 174:701-707.

(iii) Development of new methods for isolation and characterization:Lange and Vallee, 1976, "Double-Ternary Complex Affinity Chromatography:Preparation of Alcohol Dehydrogenases," Biochemistry 15:4681-4686; Keunget al., 1985, "Identification of Human Alcohol Dehydrogenase Isozymes byDisc Polyacrylamide Gel Electrophoresis in 7M Urea," Biochem. Biophys.Res. Comm. 151:92-96; Montavon et al., 1989, "A Human Liver AlcoholDehydrogenase Enzyme-Linked Immunosorbent Assay Specific for Class I,II, and III Isozymes," Anal. Biochem. 176:48-56.

(iv) Characterization of isozymes: von Wartburg et al., 1964, "HumanLiver Alcohol Dehydrogenase Kinetic and Physicochemical Properties,"Biochemistry 3:1775-1782; Blair and Vallee, 1966, supra; Lange et al.,1976, "Human Liver Alcohol Dehydrogenase: Purification, Composition, andCatalytic Features," Biochemistry 15:4687-4693; Wagner et al., 1983,"Kinetic Properties of Human Alcohol Dehydrogenase: Oxidation ofAlcohols by Class I Isoenzymes," Biochemistry 22:1857-1863; Wagner etal., 1984, "Physical and Enzymatic Properties of a Class III Isozyme ofHuman Liver Alcohol Dehydrogenase: χ-ADH," Biochemistry 23:2193-2199;Ditlow et al., 1984, "Physical and Enzymatic Properties of a Class IIAlcohol Dehydrogenase Isozyme of Human Liver: π-ADH," Biochemistry23:6363-6368; Fong and Keung(a), 1987, "Substrate Specificity of HumanClass I Alcohol Dehydrogenase Homo- and Heterodimers Containing the β₂(Oriental) Subunits," Biochem. 26:5726-5732; Fong and Keung(b), 1987,"β₂ (Oriental) Human Liver Alcohol Dehydrogenases Do Not Exhibit SubunitInteraction: Oxidation of Cyclohexanol by Homo- and Heterodimers,"Biochem. 26:5733-5738.

(v) Relationship of isozymes to genetics: Li et al., 1977, "Isolation ofAlcohol Dehydrogenase of Human Liver: Is it a Determinant ofAlcoholism?" Proc. Natl. Acad. Sci. USA 74:4378-4381; J ornvall et al.,1984, "Human Liver Alcohol Dehydrogenase: Amino Acid Substitution in theβ₂ β₂ Oriental Isozyme Explains Functional Properties, Establishes anActive Site Structure, and Parallels Mutational Exchanges in the YeastEnzyme," Proc. Natl. Acad. Sci. USA 81:3024-3028; von Bahr-Lindstr om etal., 1986, "cDNA and Protein Structure for the α Subunit of Human LiverAlcohol Dehydrogenase," Biochemistry 25:2465-2470; H oog et al., 1987,"Structure of the Class II Enzyme of Human Liver Alcohol Dehydrogenase:Combined cDNA and Protein Sequence Determination of the π Subunit,"Biochemistry 26:1926-1932; Fong et al., 1989, "Liver Alcohol andAldehyde Dehydrogenase Isozymes in a Chinese Population in Hong Kong,"Human Heredity 39:185-191.

(vi) Tissue distribution of isozymes: Par es et al., 1984, "OrganSpecific Alcohol Metabolism: Placental χ-ADH," Biochem. Biophys. Res.Comm. 119:1047-1055; Beisswenger et al., 1985, "χ-ADH is the SoleAlcohol Dehydrogenase Isozyme of Mammalian Brains: Implications andInferences," Proc. Natl. Acad. Sci. USA 82:8369-8373.

One ADH isozyme, class III or χ-ADH, is the only one present in brain,placenta, and testis and is least capable of oxidizing ethanol (Par esand Vallee, 1981, supra; Par es et al,, 1984, Supra; Beisswenger et al,,1985, supra). As a consequence, these tissues would seem to he atgreatest risk with respect to the effects of ethanol. On the other hand,this circumstance also affords these tissues protection fromacetaldehyde, the highly toxic oxidation product of ADH.

Alcohol abuse and alcoholism are problems unique to humans. It may notbe surprising, therefore, that the complexity in other species issignificantly less than in man. Such species differences extend to thecatalytic preferences of ADH isozymes toward different alcohols. Forexample, horse ADH does not oxidize methyl alcohol and ethylene glycolwhile human ADH does (von Wartburg et al., 1964, supra). Large doses ofethanol administered to compete with methanol or ethylene glycol andprevent their oxidation to toxic products now constitutes the therapyfor individuals poisoned with these agents (Wacker et al., 1965, supra).As a consequence of the detailed research exemplified above, much moreis known about human ADH than the corresponding enzyme in other species,a unique situation quite the opposite for most other enzymes.

Each of the human ADHs is composed of two protein subunits that form adimeric molecule. Class I ADHs are made up of α, β, and γ subunits whichcombine into homodimeric and heterodimeric isozymes; class II, III andIV appear to be only homodimers (Vallee and Bazzone, in Isozymes:Current Topics in Biological and Medical Research, Rattazzi et al.(eds.) pp. 219-244, Alan R. Liss, Inc., N.Y., 1983; Vallee, B. L., ANovel Approach to Human Ethanol Metabolism: Isoenzymes of AlcoholDehydrogenase. Invited Lecture, Proceedings of the 20th InternationalEuropean Brewery Convention, Helsinki, 1985; Par es et al., 1990, FEBSLett. 227:115-118). The activities of the different ADHs toward severaltypes of substrates has been examined and is quite revealing (see, forexample, Vallee, 1985, supra). Class I isozymes containing at least oneγ-subunit are active toward specific steroid hormones and areselectively inhibited by testosterone (M ardh et al., 1986a, supra;McEvily et al., 1988, Supra). Class II ADH contains the π-subunit and isthe only one that acts selectively on intermediates in the metabolism ofnorepinephrine, a critical endocrine and neurotransmitter agent (M ardhet al., 1986c, supra). The class III (χ) enzyme and its uniquecharacteristics were mentioned above. The recently discovered humanclass IV ADH (Moreno and Par es, 1991, J. Biol. Chem., 266:1128-1133),found mainly in gastric mucosa, shares the general physicochemicalproperties of all mammalian ADHs. Kinetically, it resembles class II ADHbut is chemically distinct. Since ethanol concentration in the stomachsof drinkers may be as high as 1 to 10M transiently, the moderately highK_(m), 41 mM, of this isozyme is nevertheless ample to allow it to havea possibly important role in the first pass metabolism of ethanol. Manyalcohols other than ethanol have important physiological roles and someare likely to be substrates for one or another of the ADH isozymes.Clearly, the interference of ethanol with normal metabolic processescould have serious consequences, both acute and chronic. One of the maingoals of continued research is the identification of these criticalsubstrates.

Genetic subvariants of the β and γ-subunits of ADH isozymes within thegeneral population (β₁, β₂, β₃, and γ₁, γ₂) produce characteristicdifferences in individuals. The first genetic difference found betweenthe form predominant in Caucasians (β₁) and that predominant in Asians(β₂) is also the most profound (Smith et al., 1971, Ann. Hum. Genet.,Lond., 34:251-271; Fukui and Wakasugi, 1972, Jpn. J. Leg. Med.,26:46-51); the β₁ -subunit is 100-times less effective in convertingethanol to acetaldehyde than is the β₂ -subunit. All of the differencesare now known to result from point mutations at widely differentpositions in the chain, e.g., β₁ →β₂, R47H; β₁ →β₃, R369C; γ₁ →γ₂, I349Vand R271Q but all affect coenzyme binding (J ornvall et al., 1987,Enzyme 37:5-18). Several population studies have documented strikingdifferences in β₁ and β₂ frequencies among Asian and Caucasianpopulations. For example, in an Asian population in Hong Kong, the β₁form of the γ-subunit was present in only about 10% of the subjects; allothers had the β₂ form (Fong et al., 1989, supra). In contrast, studieson a Caucasian population in England indicated that 90% had the β₁ formand only 10% had the β₂ form (Smith et al., 1971, supra).

Aldehyde dehydrogenase (ALDH) is the enzyme that catalyzes the secondstep in the ethanol metabolic pathway (see reaction formula above). Aswith ADH, there are multiple forms of ALDH, but only two of these havebeen examined in any detail; very much less is known about the others.The first two classes, in particular, are thought to have primaryresponsibility for oxidizing acetaldehyde (Pietruszko, in Biochemistryand Physiology of Substance Abuse, Watson (ed.), pp. 89-127, 1989).ALDH-I is present in mitochondria, has a high affinity for acetaldehyde,and has been assigned the major role in acetaldehyde detoxification.ALDH-II, on the other hand, occurs in the cytosol and has a low affinityfor acetaldehyde. It is therefore thought to be less effective in itsdetoxification. The amino acid sequences of both forms are now known (Jornvall et al., 1987, supra).

An important inactive dominant mutant form of ALDH-I was discovered byGoedde et al., 1979, Hum. Genet. 51:331-334, and shown to be present inapproximately 50% of major Asian populations, e.g., Chinese, Japaneseand Vietnamese (Goedde and Agarwal, 1987, Enzyme, 37:29-44). This mutantprotein apparently results from at least one point mutation (K487E)(Yoshida et al., 1984, Proc. Natl. Acad. Sci. USA 81:258-261) thatabolishes enzymatic activity and therefore markedly impairs the abilityof heterozygous and homozygous individuals (Goedde and Agarwal, 1990,Pharm. Ther., 95:345-371) to metabolize a variety of aldehydes includingacetaldehyde and presumably including any physiologically importantaldehydes that are in the range of the specificity characteristic ofnative ALDH-I. Remarkably, such individuals do not display anypathologic abnormalities but do experience a sensitivity reaction whenthey consume alcohol. The characteristic facial flushing is the symptomof this reaction that is recognizable immediately. Still moreremarkably, this mutation seems to have survival value: alcoholism andalcohol abuse virtually do not exist among Asian flushers (Ohmori etal., 1986, Prog. Neuro-Psychopharmacol. and Biol. Psychiat.,10:229-235).

The Hong Kong study (Fong et al., 1989, supra) documents, for the firsttime, the joint distribution of the β-ADH and ALDH-I genetic subvariantsin a Chinese population. The subvariants classify into four measurablydistinct subgroups: 2.2% β₁ -ADH and active ALDH-I; 5.6% β₁ -ADH andinactive ALDH-I; 44.4% β₂ -ADH and active ALDH-I; and 47.8% β₂ -ADH andinactive ALDH-I. Based on the catalytic capacities of the four phenotypevarieties, one would expect subjects with β₂ -ADH and inactive ALDH-I tobe the most rapidly intolerant of alcohol; those with β₁ -ADH andinactive ALDH-I to be intolerant of alcohol but with less rapid onset;those with β₂ -ADH and active ALDH-I to be moderately tolerant; andsubjects with β₁ -ADH and active ALDH-I, i.e., the predominant Caucasiantype, to be tolerant.

Since the lack of ALDH-I is not known to generate other significantmetabolic problems, save those which are the consequence of ethanolmetabolism, it would be ideal if a drug could be found which mimics theeffect of this natural genetic variant but without producing substantialtoxic side effects; such a drug would clearly offer great promise forthe treatment of alcoholism and alcohol abuse.

The experience of Asian flushers with alcohol is not described as"aversion," but rather as intolerance, i.e., as an inability to endurealcohol. This is an important distinction because in Western medicinethe psychological setting surrounding the administration of the toxicdrugs disulfiram and carbimide has been given considerable emphasis inproducing a regimen leading to so-called "aversion therapy" and morerecently "psychological deterrence" (Banys, 1988, J. Psychoactive Drugs26:243-261).

We now describe in detail two types of treatments for alcoholism andalcohol abuse that were known long before either the enzymology orgenetics of ADH and ALDH isozymes were known. Their discovery and usehas been phenomenological: not based on modern rational drug discoveryor design. On the one hand, Western medicine has used toxic chemicals,not further developed since discovery of their effects on exposedindustrial workers decades ago, to produce sensitization to alcohol.Ancient Traditional Chinese medicine, on the other hand, has used herbalpreparations to treat diseases generally, and in particular alcoholintoxication, according to a philosophy in which herbal mixturesmodulate bodily functions; treatment with herbal combinations is highlyindividualistic both with respect to the practitioner's preferences andprescriptions for the patient; record-keeping is rare; and practice ofthe art is heavily influenced by oral anecdotal tradition.

The only two pharmaceuticals currently used as alcohol-sensitizing drugsare both chemically reactive species but differently so, bothnon-specific inhibitors and individually distinct and hence differentfrom one another, and both shown after decades of testing and use to betoxic, unsafe and ineffective. The pharmacological basis for the actionof these drugs, disulfiram and carbimide (hereinafter referred to by itschemical name, cyanamide) is thought to be inhibition of hepatic ALDHs,but neither one is selective for ALDH-I, the only ALDH known to beaffected by genetic mutation.

Disulfiram

Disulfiram (tetraethylthiuram disulfide) was first proposed as anaversive agent for the treatment of alcoholism by Williams, 1937, JAMA109:1472-1473. He had noticed that workers in the rubber industry whohad been exposed to thiuram compounds, which are used as accelerators ofvulcanization, experienced unpleasant effects after consumption ofalcohol. Its approved use as a drug dates from 1948.

As to chemical properties, disulfiram is a general reagent for thedetermination of SH groups in proteins (Neims et al., 1966, J. Biol.Chem. 241, pp. 3036-3040), and reacts with thiols to form thediethylammonium diethyldithiocarbamates, carbon disulfide and thedisulfide derived from the thiol (Coffey, supra, pp. 331-332); itundergoes disulfide exchange reactions under mild conditions.

Given its chemical properties, it is not surprising to find thatdisulfiram is a broadly acting but non-specific inhibitor of manyphysiologically important sulfhydryl-containing compounds includingenzymes, Wright and Moore, 1990, Am. J. Medicine, 88:647-655 (for areview, see Banys, 1988, supra). Thus, it inhibits enzymes critical inneurotransmitter metabolism (dopamine-β-hydroxylase), drug metabolismand detoxification (microsomal mixed function oxidases), and multiplepathways of intermediary metabolism. It is a potent inhibitor of manyliver enzymes, including ALDH, DBH, aniline hydroxylase,nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, andcytochrome P-450. Other studies have demonstrated inhibition ofglyceraldehyde-3-phosphate dehydrogenase, succinic dehydrogenase,xanthine oxidase, hexokinase, and NADPH dehydrogenase. Still otherstudies have established inhibition of superoxide dismutase, which isthought to be an important antioxidant defense mechanism against freeradical-induced biological damage. The details of these and otherinstances of enzyme inhibition may be found in the references cited inBanys, 1988, supra. This lack of specificity clearly contributes to andmay be largely responsible for the substantial toxicity that accompaniesthe therapeutic use of disulfiram.

In vitro, disulfiram (Pietruszko, 1989, supra) is a potent inhibitor ofthe high K_(m) cytosolic isozyme (ALDH-II) but inhibits the majoracetaldehyde oxidizing mitochondrial isozyme (ALDH-I) only slightly.However, under conditions where trace amounts of certain mercaptans suchas 2-mercaptoethanol or the in vivo metabolite methanethiol are added todisulfiram to generate a mixed disulfide, the low K_(m) mitochondrialALDH-I isozyme, normally resistant to disulfiram, is inactivated. Thus,disulfiram directly inhibits ALDH-II, but only indirectly inhibitsALDH-I via metabolites (Pietruszko, 1989, supra).

In vivo, disulfiram acts slowly to inhibit ALDH over 12 hours, and thisinhibition is irreversible (Pietruszko, 1989, supra). Restoration ofALDH activity after disulfiram administration depends upon de novoenzyme synthesis of ALDH, which requires 6 or more days. Thus,disulfiram and its metabolites have the capacity to shut down hepaticacetaldehyde oxidation via ALDH-I and ALDH-II so that in the presence ofhigh concentrations of ethanol, high levels of acetaldehyde will rapidlyaccumulate. Although exogenous acetaldehyde is known to be toxic, it isnot at all clear that endogenous accumulation of acetaldehyde is theonly or even the main causative agent in the so-calleddisulfiram-alcohol reaction (DAR) described below. The directinvolvement of acetaldehyde in any of the manifestations of alcoholintolerance is poorly studied, poorly understood and remains unproven.

Disulfiram is essentially the only alcohol-sensitizing agent approvedand marketed for use in the U.S. by Wyeth-Ayerst as Antabuse® and hasbeen used in alcohol-aversion and psychological deterrence therapy. In apatient who has consumed ethanol, inhibition of ALDH by disulfiramproduces highly unpleasant physiological reactions, among them flushing,tachypnoea, palpitations, nausea and tachycardia (Peachey and Naranjo,1985, Medical Progress, May:45-59). The rationale for treatment withdisulfiram is that fear of these reactions will deter alcoholics fromfurther drinking (Peachey and Naranjo, 1985, supra).

As described in the 1991 Physician's Desk Reference (Medical EconomicsCo., Oradell, N.J., pp. 2358-59), Antabuse® plus alcohol, even smallamounts, produces flushing, throbbing in the head and neck, throbbingheadache, respiratory difficulty, nausea, copious vomiting, sweating,thirst, chest pain, palpitation, dyspnea, hyperventilation, tachycardia,hypotension, syncope, marked uneasiness, weakness, vertigo, blurredvision, and confusion (Physician's Desk Reference, 1991, supra).Significant cardiac, hepatic, and neurological toxicity, have beenobserved associated with disulfiram therapy. For example, in severereactions to Antabuse®, there may be respiratory depression,cardiovascular collapse, arrhythmias, myocardial infarction, acutecongestive heart failure, unconsciousness, convulsions, and death (seePhysician's Desk Reference, supra). These at best undesirable sideeffects have been attributed to inhibition of enzymes other than ALDHs,as well as inhibition of the normal physiological functions of one ormore of the ALDHs. In fact, the risk of taking disulfiram is so high inthe minds of many that many clinicians refuse to use this drug to dealwith alcohol abuse. Moreover, many patients themselves either refuse totake it or abandon its use. Thus, the art has not yet been provided witha drug for the selective or direct reversible inhibition of ALDH-Iwithout the undesirable side effects or toxicity which accompaniesdisulfiram treatment.

In fact, placebo-controlled clinical trials of Antabuse® (disulfiram)(Fuller et al., 1986, JAMA 256:1449-1455; Fuller and Roth, 1979, Ann.Int. Med. 90:901-904) have shown that disulfiram is no more effectivethan the placebo control in reducing alcohol consumption, when comparedwith pre-treatment levels. According to Banys, 1988, supra, althoughsince 1948 millions of doses of disulfiram have been prescribed for thetreatment of alcoholism, well-controlled studies have never demonstratedthat disulfiram is more effective than placebos in producing sustainedabstinence; most of the studies published in the ensuing 40 years sufferfrom serious flaws. In reviewing the efficacy of disulfiram, Banys,1988, supra supports the contention of Sellers et al., 1981, N. Eng. J.Med. 305:1255-1262, that "evidence supporting the efficacy of disulfiramis limited. Controlled clinical trials of efficacy show no improvementor short-term improvement only. Appreciable improvements (abstinence andimproved social functioning) reported by chronic alcoholics during thefirst three months of treatment with therapeutic doses (250 mg daily)and non-therapeutic doses (1 mg daily) probably result fromnon-specific, nonpharmacologic activity of the drug. The subsequentdecline from early improvement after the first three months of treatmentprobably reflects both the low potency of the drug and the increasedimportance of nonpharmacologic factors as determinants of long-termoutcomes of treatment.

In accord with this, of all the numerous studies of disulfiram,according to Peachey et al.(a), 1989, Brit. J. Addict. 84:877-887, onlytwo properly controlled clinical trials were conducted, and the morerecent of these two reported that disulfiram was no more effective thanplacebos in bringing about continued abstinence in alcoholic patients.

Thus, the weight of the evidence after more than fifty years of use isthat disulfiram is not only toxic and unsafe but ineffective.

Cyanamide

The citrated calcium salt of cyanamide was introduced as a result of thesearch for an alcohol-sensitizing agent less toxic than disulfiram(Ferguson, 1956, Canad. M. A. J., 74:793-795; Reilly, 1976, Lancet (Apr.24, 1976): 911-912), but even now only disulfiram has been approved foruse in the United States. Citrated calcium cyanamide is hydrolyzed tofree cyanamide (H₂ NCN) in aqueous solution, hence the generalproperties of cyanamide are relevant. Like disulfiram, cyanamide'salcohol-sensitizing effect was discovered among industrial workersexposed to the substance in the workplace. Although chemically distinctfrom disulfiram, it is also a reactive species. Cyanamide, which readilyforms compounds by addition to the cyano group, yields guanidiniumcompounds, O-alkylisoureas and S-alkylisothioureas when reacted withalkyl amines, alcohols and thiols, respectively (Rodd's Chemistry ofCarbon Compounds, 1965, Vol. 1, Part C, Coffey, ed., Elsevier,Amsterdam, p.374), i.e., with the nucleophilic functionalities that arepresent in proteins. It is so reactive that at slightly alkaline pH itdimerizes to cyanoguanidine, a species that is itself reactive towardnucleophiles, e.g., alkyl amines (Rodd, 1965, supra, p. 349).Incorporation of citrate in the pharmaceutical formulation provides theslightly acid pH required for stability with respect to dimerization.

Neither ALDH-I (the low K_(m) isozyme) nor ALDH-II (the high K_(m)isozyme) are inhibited in vitro by cyanamide, but in vivo a reactiveproduct of cyanamide catabolism inhibits both isozymes (Deitrich et al.,1976, Biochem. Pharmacol. 25:2733-2737; DeMaster et al., 1982, Biochem.Biophys. Res. Comm. 107:1333-1339). Formation of this active inhibitorwas shown initially to be catalyzed by enzyme(s) present in intactmitochondria and the microsomal fraction of rat liver (DeMaster et al.,1983, Pharmacol. Biochem. Behav. 18 (Supp. 1): 273-277). More recently,mitochondrial catalase has been shown to activate cyanamide to an ALDHinhibitor (DeMaster et al., 1984, Biochem. Biophys. Res. Comm.122:358-365; Svanas and Weiner, 1985, Biochem. Pharmacol. 34:1197-1204).Further, Shirota et al.(a), 1987, Alcohol & Alcoholism Supp. 1:219-223and Shirota et al.(b), 1987, Toxicol. Let. 37:7-12, showed thatcyanamide inhibits ALDH via a reactive species and that cyanide isgenerated as a product of cyanamide oxidation by catalase underconditions in which the ALDH inhibitory species is also generated.According to Shirota et al.(b), 1987, supra, this cyanide formationcould serve as a basis for cyanamide toxicity in vivo. It was postulatedin 1987 (Shirota et al.(b), 1987, supra) that the oxidation of cyanamidewould yield nitroxyl (HNO) as a product and that this highly reactivesubstance is the active ALDH inhibitory species. In 1990, Nagasawa etal. (J. Med. Chem. 33:3120-3122) presented evidence, via isotope tracerexperiments, that nitroxyl was formed in the catalase-mediatedbioactivation of cyanamide. They suggest that their data and those ofothers support nitroxyl as the active ALDH inhibitor, noting thatmillimolar concentrations of cyanide do not inhibit ALDH. Marchner andTottmar, 1978, Acta Pharmacol. et Toxicol. 43: 219, have reported thatinhibition of ALDH with cyanamide is maximal at 1-2 hours after drugadministration and is reversible, with restoration of 80% of the ALDHactivity occurring within 24 hours.

As with disulfiram, cyanamide has been used in alcohol-aversion andpsychological deterrence therapy as described above (Peachey andNaranjo, 1985, supra). According to Peachey, 1981, J. Clin.Psychopharmacol. 1:368-375, cyanamide has not been approved in theUnited States because of its significant antithyroid activity inexperimental animals. Citrated calcium cyanamide is marketed in othercountries as Temposil®, Dipsan® and Abstem® (Shirota et al.(a), 1987,supra). "Plain" cyanamide, commonly used in Spain, is marketed as Colme®(Val erdiz and V azquez, 1989, Appl. Pathol. 7:344-349).

Cyanamide like disulfiram is reported to be associated with medicalcomplications, again as might be expected from its chemical reactivity.Although fewer side effects have been reported with cyanamide than withdisulfiram, cyanamide has been studied much less intensively and theinformation on this drug, including its side effects, especially thosewhich are long-term, is incomplete.

There are a number of known contraindications to treatment withcyanamide. Among the toxic effects of cyanamide reported are thefollowing: (i) allergic contact dermatitis according to Conde-Salazar etal., 1981, Contact Dermatitis 7:329-330 and references cited therein andperipheral neuropathy (also associated with disulfiram) according toReilly, 1976, supra, who suggests that both cyanamide and disulfiram aregeneral metabolic poisons and may lead to the accumulation of toxicderivatives of chemicals normally metabolized by oxidative pathways;(ii) liver injury, including generation of ground-glass inclusion bodiesin liver cells of alcoholics treated with cyanamide (but not disulfiram,V azquez et al.(a), 1983, Diagnostic Histopath. 6:29-37) as firstreported by V azquez and Cervera, 1980, Lancet 1:361-362 using plaincyanamide and by Thomsen and Reinicke, 1981, Liver 1:67-73 as well asKoyama et al., 1984, Acta Hepatol. Jpn. 25:251-256 using the citratedcalcium salt of cyanamide; a series of reports of hepatotoxicity,including ground-glass inclusions, inflammatory reactions associatedwith liver cell destruction, portal tract fibrosis that can be severe iftreatment has been prolonged, scarring, even cirrhosis according to theabove-cited references and V azquez et al.(a), 1983, supra; V azquez etal.(b), 1983, Liver 3:225-230; Bruguera et al., 1986, Arch. Pathol. Lab.Med. 110:906-910; Bruguera et al., 1987, Liver 7:216-222; Val erdiz andV azquez, 1989, supra, for cyanamide and disulfiram but not calciumcyanamide; and (iii) cardiotoxic effects, including hypotension and evencardiac death according to Rodger, 1962, Br. Med. J. 2:989 and hazardouscardioacceleration according to Kupari et al., 1982, J. Toxicol. - Clin.Toxicol. 19:79-86; Kupari et al., 1982, supra suggest that the use ofalcohol aversive drugs including disulfiram and cyanamide has beencontraindicated to patients with known cardiac diseases, but point outthat it is common that asymptomatic chronic alcoholics have a number ofcardiac problems. Clearly, therefore such drugs may be hazardous.

Peachey et al.(b), 1989, Brit. J. Addict. 84:1359-1366, have conductedthe only placebo-controlled, double-blind clinical trial of Temposil®.From this short-term trial, Peachey and his colleagues concluded thatthis drug was safe for use in alcoholics with normal thyroid functionand without other serious medical conditions. Thyroid function was notaltered during the short-term trial by Temposil® in patients with normalpretreatment thyroid function. However, in the trial one patient whosebaseline thyroid function was decreased became hypothyroid afteradministration of Temposil®; thus it was concluded that for short-termuse in alcoholics with normal thyroid function, the drug was safe.Peachey et al.(a), 1989, supra, report that they did not observehepatotoxicity as measured merely by blood alkaline phosphatase. Liverbiopsies were not performed, so that an assessment of histopathologicalliver changes in biopsies, such as those cited above with reference tohepatoxicity of cyanamide, was not done. Despite the prematureconclusion of safety by Peachey et al.(a), 1989, supra, as limited bytheir assessment of what was measured as short-term effects, the effectsof long-term treatment with cyanamide in controlled studies is stillunknown.

According to Peachey, 1981, supra, in Canada and other countries,cyanamide has not been used widely because of its short duration ofactivity and its questionable efficacy in reducing drinking.Unfortunately, placebo-controlled clinical trials of Temposil® (chemicalname: calcium cyanamide; generic name: calcium carbimide) (Peachey etal.(a), 1989, supra; Peachey et al.(b), 1989, supra) have shown that,compared with pre-treatment levels, cyanamide is only as effective asthe placebo control in reducing alcohol consumption.

The weight of the evidence is that cyanamide in its various forms, likedisulfiram, is not only toxic and unsafe but ineffective.

There are some reports that use of either disulfiram or cyanamide iscounterproductive in treatment of alcoholism. In a double-blind study inhumans, consumption of low doses of alcohol together with eitherdisulfiram or cyanamide, induces and enhances euphoria (Brown et al.,1983, Alcoholism: Clin. Exp. Res. 7:276-278). Brien et al., 1980, Eur.J. Clin. Pharmacol. 18:199-205, have reported that their results withmale alcoholic volunteers ingesting small amounts of ethanol after oraladministration of cyanamide support the self-reports of alcoholics whostate that they can circumvent a severe disulfiram-ethanol reaction byingesting ethanol over a few hours, and thereafter drink excessivelywith impunity, the so-called burn-off phenomenon. If both disulfiram andcyanamide can be effectively burned-off by slow ingestion for a periodfollowed by excessive consumption without aversion, the effectiveness ofthese so-called anti-alcohol drugs not only may be severely limited buteven generally counterproductive.

Cyanamide has also been shown to have the undesirable effect of actuallycausing an increase in alcohol consumption in animals given cyanamideafter alcohol deprivation (Sinclair and Gribble, 1985, Alcohol7:627-630). Typically cyanamide is given to alcoholics after they havebeen withdrawn from alcohol and are being abstinent. According toSinclair and Gribble, 1985, supra, if this results in a potentiation ofthe desire for alcohol subsequent to termination of the drug, as appearsto be the case in rat experiments, treatment with cyanamide would becounterproductive and should be dropped from usage altogether.

Traditional Chinese Herbal Medicine

Since ancient times, Radix Puerariae (RP), prepared from the root ofPueraria lobata Ohwi or Pueraria pseudo-hirsuta Tang et Wang(Leguminosae) and Puerariae Flos (FP), prepared from the flower ofPueraria lobata Ohwi have been known for their use in TraditionalChinese medicine. The crude drug RP was described in the first ChineseMateria Medica about 200 B.C. as something of a panacea: an antipyretic,antidiarrhetic, diaphoretic, anti-emetic agent, and, in today'sparlance, a general anti-microbial agent. S un S imi ao reported the useof RP for the relief of drunkenness in his work "B eij i-Qi anj in-Y aofang" about 600 A.D. Presently, RP is widely used by the Chinese for thetreatment of drunkenness, muscle clonus and tonus and myalgia,hypertension, migraine, angina, arrhythmia, and febrile diseases ingeneral (Qu angu o Zh ongc aoy ao Hu ibi an editing group, pp. 829-830,Qu angu o Zh ongc aoy ao Hu ibi an People's Health Publisher, Beijing,1983). It has been applied also to treat symptoms of febrile illnessincluding chills, and is administered as a root decoction, whoseprincipal use was based on its diaphoretic, antipyretic and spasmolyticeffects, according to Niiho et al., 1989, Yakugaku Zasshi 109:424-431(English translation). According to Niiho et al., 1989, supra, RP isprescribed as a flower decoction to "activate the stomach, stop thethirst and relieve alcohol intoxication," and is believed to have aneffect on alcohol elimination.

Although RP has been a part of Chinese medical practice for more than2000 years, only in the past several decades have attempts been made topurify and classify its active ingredients (see, for example, F ang,1980, J. Ethnopharmacol. 2:57-63 and references cited therein, includingF ang et al., 1974, Zh ong Hu a Y i Xu e Z a Zh i (Chinese MedicalJournal) 5:271-274; Ch en and Zh ang, 1985, Zh ong Y ao T ong B ao10:34-36; Shibata, 1979, Amer. J. Chin. Med. 1:103-141).

RP is a complex mixture with a multiplicity of components, only some fewof which have been identified. Besides starch major constituents includedaidzein, daidzin, puerarin, genistein, 6,7-dimethoxycoumarin,formononetin, β-sitosterol, allantoin and 5-methylhydantoin. The onlypharmacological activities of crude RP which have been studied are itseffects on smooth muscle and cerebrovascular and cardiovascular systems.In this regard puerarin is the primary active constituent examined forthis purpose (for a review, see L ai and T ang, 1989, Zh ong Gu o Zh ongY aao Z a Zh i 14:308-311; see also, F ang, 1980, supra).

Daidzein has been examined regarding its metabolic fate, but not withregard to any human pharmacological effectiveness, disease state or bodysystem. It is metabolized rapidly, with a half-life on the order of onehour, after intravenous administration to mice (Yueh and Chu, 1977,Scientia Sinica 20:513-521; S u and Zh u, 1979, Acta PharmaceuticalSinica 14:134 (Abstract)); an experiment in which daidzein wasadministered to two human volunteers revealed only that little daidzeinhad appeared in urine and feces after 60 hours. With this exception, themetabolic fate of daidzein in humans remains unknown. Similarly there isvery little knowledge about the effects of crude RP or its constituentson acute or chronic alcohol intoxication.

With respect to daidzin, the only reported pharmacological activity isits estrogenic activity at high doses (Farmakalidis and Murphy, 1984,Fd. Chem. Toxic. 22:237-239; Price and Fenwick, 1985, Food Add. Contam.2:73-106); daidzin administered subcutaneously in propylene glycolshowed no antifebrile (hypothermic) effect in rats and showed nospasmolytic effect in mice (Nakamoto et al., 1977, Yakugaku Zasshi97:103-105). Thus, the art has not yet identified any components of RPor their activities in the metabolism of ethanol and/or the mediation ofthe behavioral effects of ethanol. Further, to increase ethanolelimination, RP has been employed in Traditional Chinese medicine inorder to relieve or remedy excess alcohol consumption. With respect toethanol metabolism via ADH and ALDH, this would suggest that componentsof RP would activate, not inhibit, ADH and ALDH to eliminate consumedethanol more rapidly. Unexpectedly, we have found ADH-inhibitorycompounds in RP. Such compounds, and methods for their use in thetreatment of drug-alcohol reactions, have been described and claimed byus in co-pending and co-assigned U.S. application Ser. Nos. 07/724,213and 07/723,945 filed Jul. 1, 1991, hereby incorporated by reference intheir entirety.

In the present invention, a hitherto completely unknown inhibitor ofALDH has been unexpectedly identified and purified from RP. Thisinhibitor is daidzin, a compound which selectively inhibits the activityof ALDH-I. Daidzin is a potent, yet reversible, inhibitor of ALDH-I, theenzyme whose mutation and resultant inactivation in about 50% of allChinese, Japanese, Vietnamese and yet other Orientals results in theiravoidance of ethanol and correlates with the virtual non-existence ofalcoholism in this group (Ohmori et al., 1986, supra). Hence it isuseful in the treatment and prevention of alcoholism and alcohol abuse.Daidzin's activity mimics the effect of the naturally occurring ALDH-Igenetic variant found among the Chinese. Daidzin selectively inhibitsthe low K_(m) ALDH isozyme, hence in its presence high levels ofacetaldehyde are likely oxidized via the high K_(m) isozyme (ALDH-II).This suggests that in the presence of daidzin the accumulation ofacetaldehyde will be limited to non-toxic levels by ALDH-II, in contrastto the high levels of acetaldehyde that accumulate with disulfiram whichinhibits both ALDH-I and ALDH-II. RP, from which daidzin was isolated,has been used safely and effectively in Traditional Chinese Medicine fortwo thousand years in a number of medical conditions. Jointly thesefacts suggest that daidzin would be a direct, safe, effective andreversible agent to induce alcohol intolerance, but without significanttoxic side effects which have been consistently observed in thetreatment of alcohol abuse with the chemically-reactive and toxicdisulfiram and cyanamide. Daidzin's properties as a selective,reversible and potent inhibitor of ALDH-I, while unexpected, arevirtually ideal for a compound intended to promote alcohol intoleranceand avoidance of its abuse, as is observed in the genetic conditionwhich its use mimics. Even structurally closely related chemicalcompounds failed to mimic daidzin's selectivity for ALDH-I andremarkable potency as an ALDH inhibitor (see Tables IV and V). Prunetinand genistin were the only other naturally-occurring compounds of thosetested which selectively inhibited ALDH-I but was nearly an order ofmagnitude less potent as an inhibitor than daidzin. In fact, daidzin isthe first reversible inhibitor of any ALDH described so far with suchhigh effectiveness and selectivity. Identification of other compoundsthat may act in concert with daidzin or modify daidzin such that itsbioavailability is increased in vivo would be particularly advantageous.Bioavailability refers to the in vivo availability of a compound (e.g.,to effect its intended function, for example, as an inhibitor orsuppressor of drinking behavior) and can be measured in a variety ofways, for example, by quantitating the amount of compound circulating inthe blood relative to the amount administered. For daidzin, thebioavailability has been unexpectedly found to be increased by a factorin RP. The bioavailability of an ALDH-inhibitory analog of daidzin bysuch factor may be similarly increased.

Daidzein, the aglycone of daidzin which is also present in RP, not onlydoes not inhibit ALDH but instead selectively inhibits certain ADHisozymes. Hence, daidzein inhibits the first but not the second step inhuman ethanol metabolism, while daidzin inhibits the second but not thefirst step in human ethanol metabolism as described above. It cannot bepredicted so far on strict structural or other grounds whichflavone/isoflavone compound present in RP, or any closely relatedcompound, will inhibit ADH or a selective isozyme of ADH, ALDH or aselective isozyme of ALDH, both ADH and ALDH, or neither ADH or ALDH.For example, improved inhibitory compounds may be obtained by syntheticderivatives (i.e., analogs) of daidzin, wherein the glucose is replacedwith a different sugar moiety. For example, L and D aldo- orketo-tetroses, pentoses, hexoses, heptoses or the amino, alcohol and/oracid derivatives of such tetroses, pentoses, hexoses or heptoses; orwherein the glucose is replaced by the deoxy analogs of such tetroses,pentoses, hexoses or heptoses. Alternatively, the glucose (GlcO) moietyof daidzin may be replaced by alkoxy or acyloxy groups at the 7-positionbearing various chain lengths, for example, up to 11 or more, comprisingany of straight chain alkyl, peptidic, polyether, etc. backbones, andthe backbones may be substituted with various neutral (e.g., hydroxyl,sugar, etc.) or charged (e.g., carboxylate, phosphate, phosphonate,sulfate, sulfonate, etc.) moieties. Additionally suitable moieties(e.g., carboxylate, hydroxyl, etc.) may be esterified.

These examples are not exhaustive in scope but suggest to those skilledin the art routes to the identification of daidzin derivatives (i.e.,analogs) having increased bioavailability and improved potency,selectivity, controlled release, solubility, absorbability and/orstability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of a BioGel P-4 elution profile of RP extract.Fractions containing ALDH inhibitory materials, indicated by thehorizontal bar, were pooled and lyophilized.

FIG. 2 is a HPLC chromatogram of an ALDH inhibitor partially purified ona BioGel P-4 column (FIG. 1). Fractions containing ALDH inhibitorymaterial, indicated by the horizontal bar, were pooled and dried.

FIG. 3 is a graph of a HPLC elution profile of the ALDH inhibitorymaterial obtained in FIG. 2.

FIG. 4 is a graph of a HPLC elution profile of human ALDH activityeluted from an AMP-agarose column. Fractions containing ALDH-I andALDH-II activities were pooled and concentrated.

FIG. 5 is a picture of a starch gel electrophoretogram of ALDH-I andALDH-II preparations shown in FIG. 4.

FIG. 6 is the HPLC chromatogram of the semi-purified ALDH-I of FIG. 4.Fractions containing ALDH-I activity, indicated by the horizontal bar,were pooled and concentrated.

FIG. 7 is the HPLC chromatogram of the semi-purified ALDH-II of FIG. 4.Fractions containing ALDH-II activity, indicated by the horizontal bar,were pooled and concentrated.

FIG. 8(A) is a histogram showing the effect of Radix Puerariae (RP)extract on the total fluid intake by golden hamsters.

FIG. 8(B) is a histogram showing the effect of Radix Puerariae (RP)extract on free choice ethanol intake in golden hamsters.

FIG. 9(A) is a histogram showing the effect of daidzin on free choiceethanol intake in golden hamster-6.

FIG. 9(B) is a histogram showing the effect of daidzin on free choiceethanol intake in golden hamster-9.

FIG. 10 is a graph showing the effect of varying doses (mg/day) of crudeRP extract (solid squares) or pure synthetic daidzin (solid circles) onfree choice ethanol intake in golden hamsters, as measured by percentsuppressive response.

FIG. 11 is a graph showing the relative bioavailability (AUC) of varyingdoses (mg/animal) of daidzin administered as a crude RP extract (solidcircles) or as pure daidzin (solid squares) in a golden hamster. AUC isa measure of relative bioavailability and quantitated as the area underthe curve of the daidzin component present in plasma from a blood sampleas measured by HPLC; this correlates with the actual amount of daidzinadministered.

FIG. 12 is a graph showing the correlation between the (i) relativebioavailability (AUC, arbitrary unit) of varying doses of daidzinadministered as a crude RP extract (solid squares) or as pure syntheticdaidzin (solid circles) and (ii) the percent ethanol intake suppressionresponse in a golden hamster.

FIG. 13 is a graph showing the plasma drug concentration (inmicromoles/liter) versus time (in minutes) for a single 10 mg dosesynthetic daidzin (solid circles) or daidzein 7-(ω-carboxyhexyl) ether(designated "hepzein", solid squares) administered to a golden hamster.

SUMMARY OF THE INVENTION

The invention relates to the inhibition of aldehyde dehydrogenase, theenzyme system which is responsible for the second step in the majorpathway of ethanol metabolism in humans, using daidzin or a similarALDH-inhibiting compound, such as a synthetic analog of daidzin. In thefirst step, alcohol dehydrogenase (ADH) isozymes catalyze the conversionof ethanol to acetaldehyde. In the second step, aldehyde dehydrogenase(ALDH) isozymes catalyze the conversion of acetaldehyde to acetate. NAD⁺is a cofactor in both steps.

More particularly, the invention relates to a method for inhibiting ALDHactivity using daidzin or daidzin analog as the inhibitor. Daidzin hasbeen unexpectedly found to be a hitherto unknown, direct, highly potentyet selective, inhibitor of ALDH-I; the inhibition is reversible. Infact it is the first such inhibitor of any ALDH described so far.Daidzin analogs have been prepared which exhibit similar direct, highlypotent, but somewhat less selective, ALDH-inhibitory properties, Assuch, daidzin is useful in a method for the treatment of alcoholdependence (i.e., alcoholism) or alcohol abuse. It is also useful in amethod of alcohol sensitization. Daidzin is useful in a pharmaceuticalcomposition for inducing alcohol intolerance in humans. The existingdrug disulfiram, said to be alcohol-sensitizing or anti-alcohol,directly inhibits ALDH-II but not ALDH-I, has numerous toxic sideeffects and has been said widely to be ineffective. Nevertheless,disulfiram is the only alcohol-sensitizing drug currently approved foruse in the U.S. (Antabuse®). Disulfiram is a highly-reactive chemicalspecies which inhibits ALDH irreversibly, and in addition, inhibitsother non-ALDH enzyme systems in neurotransmitter metabolism, drugmetabolism and detoxification, and multiple pathways of intermediarymetabolism. This lack of specificity clearly contributes to and probablyis the basis of the toxicity that accompanies its use. The otherexisting alcohol-sensitizing drug, not approved for use in the U.S., iscyanamide, which is also a highly reactive chemical species. Cyanamideis unable to inhibit ALDH isozymes directly in vitro but must bebioactivated in vivo to an ALDH-inhibiting species. This species appearsto inhibit both ALDH-I and ALDH-II. The inhibition of ALDH via cyanamideis reported to be reversible and the activity to be of short duration.In contrast to both disulfiram and cyanamide, the present inventionprovides daidzin a new inhibitor which directly, selectively andreversibly inhibits ALDH-I activity. The present invention additionallyprovides daidzin analogs as ALDH-I inhibitors.

The invention encompasses compounds of the formula: ##STR1## wherein:

R represents

straight chain alkyl having 1-11 carbon atoms, or

branched chain alkyl having 1-30 carbon atoms, where the branched chainalkyl comprises a straight chain alkyl portion having 1-11 carbon atomssubstituted with straight or branched chain lower alkyl groups having1-6 carbon atoms;

hydroxyalkyl where the alkyl portion is straight chain alkyl having 2-11carbon atoms, or

branched chain alkyl having 2-30 carbon atoms, where the branched chainalkyl comprises a straight chain alkyl portion having 2-11 carbon atomssubstituted with straight or branched chain lower alkyl groups having1-6 carbon atoms;

carboxyalkyl where the alkyl portion is straight chain alkyl having 2-11carbon atoms, or

branched chain alkyl having 2-30 carbon atoms, where the branched chainalkyl comprises a straight chain alkyl portion having 2-11 carbon atomssubstituted with straight or branched chain lower alkyl groups having1-6 carbon atoms; or ##STR2## where X is straight chain alkylene having2-11 carbon atoms, or

branched chain alkylene having 2-30 carbon atoms, where the branchedchain alkylene comprise a straight chain alkylene portion having 2-11carbon atoms substituted with straight or branched chain lower alkylgroups having 1-6 carbon atoms; and

R' is straight or branched alkyl having 1-6 carbon atoms.

The invention also encompasses compounds of the formula: ##STR3##wherein:

R represents

straight or branched chain alkyl having 1-11 carbon atoms;

hydroxyalkyl where the alkyl portion is straight or branched alkylhaving 2-11 carbon atoms; carboxyalkyl where the alkyl portion isstraight or branched alkyl having 2-11 carbon atoms; or ##STR4## where Xis straight or branched chain alkylene having 2-11 carbon atoms; and

R' is straight or branched alkyl having 1-6 carbon atoms.

By lower alkyl in the present invention is meant straight or branchedchain alkyl groups having 1-6 carbon atoms, such as, for example,methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl,2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and3-methylpentyl,

By alkyl in the present invention is meant (i) straight chain alkylgroups having 1-11 carbon atoms, such as, for example, methyl, ethyl,propyl, n-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, andundecyl, or (ii) branched chain alkyl groups having 1-30 carbon atomscomprising a straight chain alkyl portion having 1-11 carbon atomssubstituted with straight or branched chain lower alkyl groups having1-6 carbon atoms. Examples of such branched chain alkyl groups are4-n-butyl-undecane, 5-ethylnonane, 4-ethyl-5-isobutyl-5-methyldecane,3-propyl-4-ethyloctane, and 4-isooctyl-3-propylundecane.

By hydroxyalkyl is meant an alkyl group substituted with a hydroxymoiety at any available position of the alkyl group. Representativehydroxyalkyl groups are, for example, hydroxyethyl, hydroxymethyl,hydroxyhexyl, hydroxypentyl, and hydroxydecyl. In addition, the alkylgroups may be substituted with more than one hydroxy moiety, i.e., thehydroxyalkyl may be a polyhydroxyalkyl group.

By carboxyalkyl is meant radicals of the structure:

where Y represents straight or branched chain alkylene having 1-11carbon atoms.

In addition the alkyl groups may be substituted with more than onecarboxy moiety, i.e., the carboxyalkyl may be a polycarboxyalkyl group.

Furthermore, the alkyl groups may be substituted with one or morehydroxy substituents and one or more carboxy substituents. The hydroxyand carboxy substituents may also be esterified using, respectively,short chain (1-6 carbon atoms) acids and alcohols.

The inhibitor, daidzin, was isolated and purified from Radix Puerariae(RP), a dried root of Pueraria lobata which has been in use inTraditional Chinese Herbal Medicine without any reports of toxic sideeffects for more than two millennia. Daidzin's inhibitory activitymimics the effect of a natural genetic mutation of ALDH-I whose effectis inactive ALDH-I. It is observed in 50% of all Chinese, Japanese,Vietnamese and others (Goedde and Agarwal, 1987, supra). These factssuggest that treatment with daidzin is a safe, effective and reversiblemeans to achieve alcohol intolerance, without the significant toxic sideeffects associated with known alcohol-sensitizing drugs.

It should be emphasized that all treatment, in the case of RP for twomillennia, in the case of disulfiram for 43 and cyanamide for 35 yearswas empirical, phenomenological and without a known or even suspectedbiochemical, pharmacological or genetic basis. Among these agents onlyRP failed to be accompanied by any known toxicity. None of thetreatments benefited from the recent progress and current state ofrelevant scientific disciplines, i.e., in genetics, biochemistry,pharmacology or toxicology. Our isolation of daidzin and recognition ofits inhibitory ALDH-I characteristics for the first time instead makeuse of that knowledge both to recognize its biological andpharmacological properties as well as other effects and to monitor itseffectiveness by biochemical assays with ALDH-I and -II and ADH-Ithrough -IV. These properties and assays were unknown at the time thatdisulfiram and cyanamide were described or tested.

The invention was made possible by several discoveries. First, we havediscovered a previously unknown inhibitor of ALDH. We have furtherdiscovered that this inhibitor is unexpectedly potent, yet selective forALDH-I, and at the same time the inhibitory effects are direct yetreversible. No other inhibitors of similar selectivity and potency werediscovered in RP, nor in the testing of many other closely relatedchemical compounds, nor in fact among other compounds. Second, we havedemonstrated that an extract containing daidzin, as well as purifieddaidzin, in the absence of other inhibitors of ALDH, has significant invivo effects on alcohol consumption in an animal model. More recently,we have discovered that daidzin analogs, with inhibitory propertieswhich mimic daidzin, may be synthesized and used to inhibit ALDH-I. Suchdaidzin analogs are potent and even when somewhat less selective forALDH-I than daidzin, the analogs have similar significant in vivoeffects on alcohol consumption in the animal model. Additionally, wehave discovered that the bioavailability of daidzin administered as acrude extract is increased by 5-10 fold as compared with thebioavailability of daidzin administered as a purified (e.g., synthetic)compound. Therefore, the invention includes a composition of matter thatcomprises purified (e.g., synthetic) daidzin or a similarly actingdaidzin analog and a bioavailability-increasing factor or factors froman extract of RP.

Thus, daidzin or a daidzin analog with inhibitory properties which mimicdaidzin is useful in a pharmaceutical composition and in a method forextinguishing an alcohol-drinking response, and in a method forsuppressing an urge for alcohol. In addition, daidzin or a daidzinanalog with inhibitory properties which mimic daidzin is useful in apharmaceutical composition and in a method for inducing alcoholintolerance or in a method of preventing alcoholism in an individualwith a susceptibility to alcoholism or alcohol abuse or in a method forlimiting alcohol consumption in an individual whether or not geneticallypredisposed.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to inhibitors of aldehyde dehydrogenase, theenzyme system in humans that is responsible for the second step in themajor pathway of ethanol metabolism. The invention further relates tonovel synthetic inhibitory compounds that are daidzin analogs. Stillfurther, the invention relates to factors which potentiate the activityof the inhibitory compounds in vivo by significantly increasing thebioavailability of an inhibitory compound, such as daidzin. Theinvention ecompasses a method for inhibiting ALDH-I activity usingdaidzin and/or daidzin analog and/or daidzin or daidzin analog incombination with a factor or factors which increase the bioavailabilityof the daidzin or daidzin analog and pharmaceutical compositionscomprising daidzin and/or daidzin analog and/or daidzin or daidzinanalog in combination with a factor or factors which increase thebioavailability of the daidzin or daidzin analog as inhibitor.

In particular, daidzin has been unexpectedly found to be a direct,potent yet selective and reversible inhibitor of ALDH-I. As such,daidzin is particularly useful as a pharmaceutical composition inmethods for the treatment of alcoholism or alcohol abuse, for alcoholsensitization, for extinguishing an alcohol-drinking response, forsuppressing an urge for alcohol, for inducing alcohol intolerance, andfor preventing alcoholism in an individual with or withoutsusceptibility to alcoholism or alcohol abuse, or for limiting alcoholconsumption in an individual whether or not genetically predisposed.

Alcoholism (i.e., alcohol dependence) and alcohol abuse are seriouspublic health problems as described in the Seventh Special Report to theU.S. Congress on Alcohol and Health From the Secretary of Health andHuman Services January 1990, "Alcohol and Health--An Overview." Twodistinct forms of problem drinking were identified in thisreport--alcohol abuse, which is defined as involving patterns of heavyalcohol intake in nondependent persons in which health consequencesand/or impairment in social functioning are associated, and alcoholdependence (i.e., alcoholism), which is differentiated from alcoholabuse on the basis of such manifestations as craving, tolerance, andphysical dependence that result in changes in the importance of drinkingin one's life and in impairment of the ability to exercise restraintover drinking. There is no consensus on specific definitions ofalcoholism, alcoholics or the like. We view the U.S. Government reportand definitions as useful to indicate the magnitude of the problem. Thatreport, however, appears to have strayed, in the direction ofclassification without having the benefit of adequate verification bythe medical profession, away from the seminal approach of Goodwin etal., 1973, Arch. Gen. Psychiatry, 28:238-243, in which a genetic link toalcoholism is demonstrated with Danish adoptees where one parent had ahospital diagnosis of alcoholism. We prefer to use a concise paraphraseof the more extensive criteria for drinking categories of Goodwin etal., 1973, supra: non-alcoholic drinkers display at most occasional andilltimed drunkenness; those afflicted with alcoholism display excessiveethanol consumption exceeding dietary and caloric needs or norms whichis consequently detrimental to interpersonal, economic and professionaleffectiveness. Thus, the development of safe and effective drugs for thetreatment of alcohol abuse and alcohol dependence are urgently needed tohelp to solve these serious health problems which are world wide.

According to the present invention, daidzin was isolated and purifiedfrom Radix Puerariae (RP), a dried root of Pueraria lobata which hasbeen used safely for 2 millennia in Chinese herbal medicine. Recently, agenetic mutation in ALDH-I has been identified in a subpopulation ofOriental individuals which results in an ALDH-I isozyme that has littleor no activity. In the ALDH-I deficient population, alcoholism andalcohol abuse virtually do not exist. The effect of daidzin's direct,potent inhibition of ALDH-I mimics the effect of thisnaturally-occurring ALDH-I genetic mutation. As shown by thepurification scheme described in Example 1, no other inhibitors ofsimilar selectivity and potency were discovered in the extract of RP,which contains a multiplicity of chemical components, only few of whichhave been previously identified. While daidzin is a known component ofRP, prior to the present invention, the art has not been provided with areason for and the identification of activity of components of RPinvolved in and responsible for effects on the metabolism of ethanol inhumans and/or the mediation of the behavioral effects of ethanol by thatmeans. Further RP has been used in Traditional Chinese medicine for thetreatment of excess alcohol consumption and as an agent to increaseethanol elimination. Therefore, it was expected that RP would containcomponents that activated ALDH (and ADH) rather than components thatwould inhibit isozyme activity. Such activation would be expected sinceRP is administered by Chinese herbalists to help eliminate the consumedalcohol more quickly. Information from China indicates that administeredRP increases metabolic rate and elimination, induces intenseperspiration as one aspect of the increased elimination, and has theeffect of accelerating the return to sobriety after acute intoxication,without the fear of use that has been associated with the administrationof disulfiram or cyanamide.

As shown in Example 2, highly purified human ALDH-I and ALDH-II isozymeswere obtained and used in a series of assays to determine the inhibitoryactivity of a variety of compounds, including some components of RP.

As shown in Example 3, numerous compounds, including other components ofRP, as well as compounds structurally related to daidzin, were testedbut were not found to have the direct, potent yet selective, inhibitoryeffect on human ALDH-I. The only other compounds tested that showed aselective inhibitory effect on ALDH-I were prunetin and genistin,however, their potency as inhibitors were approximately 10-fold lessthan that of daidzin. ALDH-I is thought to be the major isozyme inacetaldehyde detoxification due to its high affinity (low K_(m)) foracetaldehyde. A large proportion of the compounds examined forinhibitory effect were benzo[b]pyran derivatives and are eitherchromones, isoflavones or coumarins. Thus, chromone is4-H-benzo[b]pyran-4-one; coumarin is 2H-benzo[b]pyran-2-one; flavone is2-phenylchromone and isoflavone is 3-phenylchromone. Structures I, IIand III, shown as follows with ring numbering, illustrate chromones,coumarins and isoflavones, respectively, and should be used inevaluating the data in Tables IV and V of Example 3: ##STR5## Examplesof reduced pyran rings are included. In flavans and isoflavans, thepyran ring of the chromone moiety is fully saturated at the 2,3 and 4positions. The numbering systems which these compounds have in commonallows facile tabular comparison of substituent effects (see Tables IVand V of Example 3).

As shown in Examples 4, 5, 6, and 7, daidzin present in an RP extract oras a purified component from the extract, has significant in vivoeffects on alcohol consumption. These experiments were designed to testthe effect of daidzin on free choice ethanol intake in golden hamsters.Initially, an acclimation period and a pretreatment period were used toestablish an ethanol/water preference ratio, as well as an overallpattern of consistent fluid intake. After the animals received daidzinin the extract or purified from the extract, the ethanol/waterpreference ratio decreased dramatically, indicating that daidzin waseffective in what may be considered alcohol intolerance therapy. Thebioavailability of daidzin administered as a crude extract issignificantly increased (e.g., 5-10 fold as shown in Example 7) ascompared with the bioavailability of daidzin administered as a purified(e.g., synthetic) compound. Daidzin is thus useful in a pharmaceuticalcomposition to inhibit ALDH-I. Pharmaceutical compositions comprising anALDH-I inhibitory compound, such as daidzin and to a lesser extentprunetin and genistin, are useful in methods for alcohol intolerance andin methods for the treatment of alcoholism or alcohol abuse.

As shown in Examples 8, 9 and 10, daidzin analogs with inhibitoryproperties which mimic daidzin, were synthesized and used in experimentsin vitro and in vivo similar to those experiments described in Examples3-7 using daidzin. Daidzin analogs according to the present inventionwere potent ALDH-I inhibitors and even if somewhat less selective forALDH-I than daidzin, these analogs were found to have significant invivo effects on alcohol consumption in an animal model, similar todaidzin.

An ALDH-I inhibitory compound according to the present invention may beadministered orally, parenterally, by inhalation or spray or rectally indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition, there is provided a pharmaceutical formulation comprising anALDH-I inhibitory compound according to the present invention and apharmaceutically acceptable carrier. An ALDH-I inhibitory compoundaccording to the present invention may be present in association withone or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants and if desired other active ingredients. Thepharmaceutical compositions containing an ALDH-I inhibitory compoundaccording to the present invention may be in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, or syrups or elixirs. Oral administration is a highlypreferred route of administration using an ALDH-I inhibitory compoundaccording to the present invention.

In addition to the use of conventional forms of drug administration asoutlined above, a number of novel drug delivery approaches have beendeveloped as described by Langer, 1990, Science 249:1527-1533, which maybe used to administer an ALDH-I inhibitory compound according to thepresent invention. These approaches for drug delivery include drugmodification by chemical means, drug entrapment in small vesicles thatare injected into the blood stream and drug entrapment within pumps orpolymeric materials that are placed in desired bodily compartments, forexample, beneath the skin, or transdermal delivery, for example via skinpatches.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. A slow-releaseformulation of an ALDH-I inhibitory compound according to the presentinvention may enhance effectiveness of the compound.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagincus suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

An ALDH-I inhibitory compound according to the present invention mayalso be administered in the form of suppositories for rectaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials arecocoa butter and polyethylene glycols.

An ALDH-I inhibitory compound according to the present invention may beadministered parenterally in a sterile medium. The drug, depending onthe vehicle and concentrations used, can either be suspended ordissolved in the vehicle. Advantageously, adjuvants such as localanesthetics, preservatives and buffering agents can be dissolved in thevehicle.

Finally, an ALDH-I inhibitory compound according to the presentinvention may be administered as an implant.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (from about 1.0 mg to about 50.0 g per 70 kgpatient per day). The amount of active inhibitor that may be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. Dosage unit forms will generally contain between fromabout 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for anyparticular individual will depend upon a variety of factors includingthe activity of the ALDH-I inhibitor, the age, body weight, generalphysical and mental health, genetic factors, environmental influences,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular problemundergoing treatment or therapy. For example, the dose level useful forinducing alcohol intolerance may vary among individuals depending on theseverity of their alcohol abuse problem. Similarly, the dose level forsuppressing an urge for alcohol may vary among individuals, depending onthe severity of the individual's alcoholism symptoms. Further, the doselevel for preventing alcoholism in an individual with a susceptibilityto alcoholism or alcohol abuse may vary depending on the causativefactors of the susceptibility as well as the severity of thepredisposition.

EXAMPLE 1

Isolation of ALDH Inhibitors

The crude drug Radix Puerariae (RP) prepared as the dried root ofPueraria lobata was purchased from a local herbal medicine store,Vinh-Kan Ginseng Co., Boston, Mass. The crude drug was prepared andpackaged by South Project Chinese Herbs Factory, Shenzhen, Kwang Tong,The People's Republic of China and was distributed by South ProjectLtd., 37 Ko Shing St., Block C, 2/F, Hong Kong. RP may also be purchasedfrom other herbal medicine stores, for example, the Lee-Yuen-CheongHerbal Medicine Store in Hong Kong.

An ALDH inhibitor, later identified as the isoflavone daidzin, wasisolated from a methanol extract of RP by chromatography on BioGel P-4and reverse phase HPLC columns. Specifically, daidzin was isolated fromRP according to the following scheme: ##STR6##

In the first step of the above scheme, dried RP, 10 g, was ground to apowder in a domestic food processor and extracted with 100 ml ofmethanol for 10 hours in a Soxhlet extractor equipped with an all-glassextraction thimble (Kontes, Vineland, N.J.). In the next step, methanolwas removed from the extract by vacuum evaporation and the resultantsyrup was dissolved in 5 ml of 10 mM sodium phosphate, pH 7.5. In thefollowing step, undissolved materials were removed by centrifugation ina Sorvall RC5B superspeed centrifuge (10,000 rpm, 30 minutes) with aSS-34 rotor (DuPont, Wilmington, Del.). Then, the supernatant solutionwas applied to a BioGel P-4 (BioRad Laboratories, Richmond, Calif.)column (3.5×55 cm) equilibrated with the same buffer. The column waseluted at 55 ml/hour and fractions of 11 ml were collected. Arepresentative elution diagram as measured by absorbance at 214 nm isshown in FIG. 1 (solid circles). Those fractions, shown in FIG. 1 asopen circles, that exhibited ALDH inhibitory activity (assayed asdescribed below) were pooled, lyophilized and redissolved in 15%methanol in water. Next, the solution was filtered (Millipore Millexfilter, 0.45μ) and injected onto a HPLC column (Waters, Milford, Mass.;NovaPak, C18 column, 6-8μ, 7.8 mm×30 cm). The column was eluted at 2ml/minute with 15% methanol/water. A representative elution pattern isshown in FIG. 2; absorbance at 214 nm over time in minutes is shown as asolid line, while percent methanol in the gradient over time in minutesis shown as a broken line. The ALDH inhibitor eluted at about 87 minutes(FIG. 2). This inhibitory material was rechromatographed on the samecolumn pre-equilibrated with and eluted with 20% methanol/water to yielda single highly purified substance as shown in FIG. 3. A furtherrechromatography under the latter conditions yielded the ALDH inhibitordaidzin as identified by four methods: (i) mass spectroscopy; (ii) NMRspectroscopy; (iii) chemical analysis; and (iv) cochromatography with anauthentic sample of daidzin (Indofine Chemical Co., Somerville, N.J.).

Mass spectroscopy was performed using a Hewlett Packard 5985B GC/MS at70 eV in an electron ionization mode. The sample of ALDH inhibitor wasintroduced with a direct insertion probe and the temperature of theprobe was raised from room temperature to 295° C. The resultant massspectrum of the ALDH inhibitor shows fragment ion peaks at m/z 118, 136and an apparent molecular ion peak at m/z 254. This spectrum isidentical to that of the authentic compound daidzein (Ganguly and Sarre,7 Feb. 1970, Chemistry and Industry, p. 201). Since daidzein has amelting point of 300° C. (Ganguly and Sarre, 1970, supra) and it doesnot inhibit ALDH (Table V), the isolated ALDH inhibitor is a derivativeof daidzein with chemical group(s) labile to the conditions under whichthe mass spectrum was obtained.

¹ H NMR spectra were acquired at 25° C. in deuterated dimethylsulfoxide,d₆ -DMSO, (Sigma Chemical Co., St. Louis, Mo.) using a 30 degreeexcitation pulse in a Varian VXR 300S NMR spectrometer operating at299.949 Megahertz. The ¹ H NMR spectrum obtained for the authenticdaidzein is consistent with that reported previously (Kitada et al.,1985, J. Chromatography 347:438-442). A tentative assignment of all the¹ H NMR signals in the NMR spectrum was made based on reference spectrain Mabry et al., 1970, In: The Systematic Identification of Flavonoids(Mabry et al., eds.), Chapter VIII, Springer-Verlag, N.Y. The reportedspectrum was identical with that of an authentic sample of daidzein [ε10.78, 7OH; 9.515, 4'OH; 8.28, H2; 7.953 (d,J=8.79), H5; 7.37(d,J=8.30), H2' and H6'; 6.925 (q,J=2.2, 8.55), H6; 6.851 (d,J=2.2), H8;6.795 (d,J=8.30), H3' and H5']. The ¹ H NMR spectrum of the ALDHinhibitor was acquired under the same conditions [ε 9.54, 4'OH; 8.38,H2; 8.036 (d,J=8.79), H5; 7.396 (d,J=8.79), H2' and H6'; 7.222(d,J=1.95), H8; 7.13 (q,J=2.44,8.79), H6; 6.804 (d,J=8.79), H3' and H5';multiplets at 5.43 (1H), 5.0-5.2 (3H) and 4.6 (1H)]. In the low fieldregion, signals for all but the 7-OH protons on daidzein were alsoobserved for the ALDH inhibitor. The lack of the 7-OH signal wasprobably not due to a rapid exchange with D₂ O because the 4'-OH protonwhich is also water-exchangeable gave rise to a strong and sharptransition at 9.54 ppm. The fact that additional signals were observedin the high field region for the ALDH inhibitor suggested that theinhibitor is a substituted daidzein. The lack of 7-OH signal suggeststhe substituent is attached to the 7-position of the daidzein aglycone.Based on the facts that the ALDH inhibitor has a melting point (seeMerck Index) and NMR spectrum (see Kitada et al., 1985, supra) similarto that reported for daidzin, the ALDH inhibitor isolated from RP istherefore daidzin, the 7-glucoside of daidzein.

To demonstrate that the ALDH inhibitor isolated from RP was daidzin, a7-glucoside of daidzein, the sample was hydrolyzed in 2N HCl for 15hours at 70° C., a condition under which glycosidic linkages are cleaved(Beeley, 1985, In: Laboratory Techniques in Biochemistry and MolecularBiology--Glycoprotein and Proteoglycan Techniques, (Burdon and vanKnippenberg, eds.), pp. 100-152, Elsevier Science Publishers B. V.,Amsterdam). Hydrolyzed samples were spotted onto three Silica Gel 60F-254 precoated TLC plates, layer thickness 0.2 mm (E. Merck, Darmstadt,Germany) and the plates were developed in three different solventsystems: (I) ethylmethyl ketone:glacial acetic acid:methanol (6:2:2)(Stahl and Kaltenbach, 1965, In: Thin-Layer Chromatography--A LaboratoryHandbook, (Stahl, ed.), pp. 461-469, Springer-Verlag, A/P N.Y.), (II)benzene:glacial acetic acid: methanol (2:2:6) (Stahl and Kaltenbach,1965, supra) and (III) formic acid:chloroform:acetone (8.5:75:16.5)(Wagner et al., 1984, In: Plant Drug Analysis, (Scott, trans.), pp.163-193, Springer-Verlag, Berlin, Heidelberg). Authentic D-glucose anddaidzein were also spotted onto the TLC plates as standards.Unhydrolyzed ALDH inhibitor was run as a control. Daidzein and ALDHinhibitor on the TLC plates were visualized by fluorescence quenchingunder short wavelength (254 nm) UV light; glucose and ALDH inhibitorwere visualized by anisaldehyde (Sigma Chemical Co., St. Louis, Mo.)spray (Stahl and Kaltenbach, 1961, J. Chromatography 5:351-355). Thefact that ALDH inhibitor can be visualized also by the anisaldehydereagent suggested that the inhibitor contained not only a daidzeinmoiety as suggested by mass spectral data but also a carbohydratecomponent.

The R_(f) values of daidzin, daidzein, glucose, ALDH inhibitor and acidhydrolyzed ALDH inhibitor obtained in different solvent systems aregiven in Table I.

                  TABLE I                                                         ______________________________________                                        R.sub.f × 100 values of glucose, daidzein, unhydrolyzed                 and acid hydrolyzed ALDH inhibitor                                                              Solvent System                                                                I     II      III                                           ______________________________________                                        Glucose             40.sup.a                                                                              73.sup.a                                                                              --                                        Daidzein            85.sup.b                                                                              96.sup.b                                                                              43.sup.b                                  ALDH Inhibitor      69.sup.a,b                                                                            91.sup.a,b                                                                             2.sup.a,b                                Acid hydrolyzed ALDH Inhibitor                                                                    40.sup.a                                                                              73.sup.a                                                                              --                                        Acid hydrolyzed ALDH Inhibitor                                                                    85.sup.b                                                                              96.sup.b                                                                              43.sup.b                                  Daidzin             69.sup.a,b                                                                            91.sup.a,b                                                                             2.sup.a,b                                ______________________________________                                         .sup.a Detected by anisaldehyde reagent                                       .sup.b Detected under UV.                                                

The unhydrolyzed ALDH inhibitor ran as a single spot with R_(f) valuesbetween those of glucose and daidzein and comigrated with an authenticsample of daidzin. Upon hydrolysis, ALDH inhibitor was cleaved into twocomponents. One component was detected under UV and had R_(f) valuesidentical to those of daidzein in all three solvent systems studied. Theother component was detected by anisaldehyde reagent and had R_(f)values identical to those of glucose in solvent system I and II. The TLCrun in solvent system III was not developed with anisaldehyde reagentbecause of high background. These results, together with results frommelting point analysis, mass and nuclear magnetic resonance spectroscopyanalysis demonstrated that the ALDH inhibitor isolated from RP wasdaidzin, a 7-glucoside of daidzein.

To monitor the purification of the ALDH inhibitor, fractions wereassayed for ALDH inhibitory activity. To determine whether a fractioncontained ALDH inhibitory activity, ALDH activities were measured in thepresence and absence of 50 μl of each fraction in our standard pH 9.5ALDH assay medium (Fong et al., 1989, supra) 0.1M in glycine-NaOH, 0.15Min KCl, 0.6 mM in NAD⁺ (Grade III, Sigma Chemical Co., St. Louis, Mo.),30 μM in acetaldehyde and preferably 5-10 nM in ALDH-I (purifiedaccording to Example 2) or a mixture of ALDH-I and ALDH-II (obtainedafter the AMP-agarose column step described in Example 2). The enzymereaction rates were measured by monitoring the production of NADH at 340nm (ε=6.22 mM⁻¹ cm⁻¹) with a Varian Cary 219 spectrophotometerthermostated at 25° C. ALDH inhibition was calculated by the followingequation: ##EQU1## where V_(o) is the enzyme reaction rate measured inthe absence of the sample fraction and where V_(i) is the enzymereaction rate measured in the presence of the 50 μl sample fraction.

EXAMPLE 2

Purification of ALDH isozymes

Human livers were obtained at autopsy within 12 hours postmortem andwere stored at -70° C. A modification of the procedure of Ikawa et al.,1983, J. Biol. Chem. 258:6282-6287, was used to purify ALDH-I andALDH-II isozymes. A Caucasian liver sample (50 g wet weight) with theusual Caucasian ALDH phenotype (both ALDH-I and ALDH-II present) washomogenized at 0° C. in 100 ml pH 6.0 buffer 15 mM in sodium phosphate,0.5 mM in EDTA, 0.5 mM in dithiothreitol (DTT; Sigma Chemical Co., St.Louis, Mo.). The homogenate was centrifuged at 4° C. in a Beckman(Beckman Instruments Inc., Irvine, Calif.) L8-M ultracentrifuge at92,000×g for 90 minutes. The clear supernatant solution was diluted to afinal volume of 200 ml with cold water and loaded at ambient temperatureonto a carboxymethyl cellulose cake (CM-52, Whatman Lab Sales, Clifton,N.J.) packed in a 9.5×4 cm sintered glass funnel and equilibrated withhomogenizing buffer. Both ALDH-I and ALDH-II eluted in the void volumeof the CM-52 cake and this fraction was immediately loaded onto a 1.5×20cm AMP-Agarose (A-3019, Sigma Chemical Co., St. Louis, Mo.) columnequilibrated with 2× homogenizing buffer in the cold room. TheAMP-column was first washed with 100 ml of column buffer and then elutedwith a 400 ml linear gradient of 0-5 mM NAD⁺ in column buffer. Fractionscontaining ALDH activity (assayed as described in Example 1) werepooled, concentrated to about 8 ml with an Amicon PM-30 membrane (AmiconDivision, W. R. Grace & Co., Danvers, Mass.) and dialyzed in the coldroom overnight against 4 liters of pH 8.0 buffer 10 mM in Tris-HCl and 1mM in DTT for further purification on HPLC.

The HPLC fractionation of ALDH isozymes was performed with a Watersgradient chromatographic system (Waters, Milford, Mass.) consisting oftwo M45 pumps, U6K injector equipped with a 2 or 10 ml injection loop,Model 482 variable wavelength UV/VIS detector, model 680 automatedgradient controller, and 740 data module. Fractionation of less than 10mg total protein was carried out at room temperature on an analyticalProtein-Pak DEAE 5PW anion exchange HPLC column (0.75×7.5 cm) (Waters,Milford, Mass.) at a flow rate of 1 ml/minute. For protein loads ofgreater than 10 mg fractionation was carried out on a semi-preparativescale version of the same column (2.15×15 cm) at 5 ml/minute. As much as150 mg protein could be loaded onto the semi-preparative column withoutan overloading problem. The dialyzed ALDH samples were filtered and wereloaded (1-10 ml) onto a column previously equilibrated with dialysisbuffer. Elution was effected with NaCl gradients in equilibration bufferas detailed below.

The AMP-Agarose column eluate (˜100 mg total protein) was first loadedonto a semi-preparative DEAE 5PW HPLC column and the ALDH-I and ALDH-IIwere resolved, as shown in FIG. 4, with the following NaCl gradient:0-20 minute, 0-75 mM, linear; 20-120 minute, 75-100 mM, linear; 120-125minute, 100-500 mM, linear; 125-145 minute, 500 mM, isocratic; 145-155minute, 500-0 mM, linear. A representative HPLC chromatograph of theALDH isozymes from the AMP-agarose column is shown in FIG. 4; the solidline shows ALDH activity in units/ml and the broken line shows the NaCl(mM) gradient as plotted against fraction number. At this stage, ALDH-Iand ALDH-II isozymes were well resolved as shown in FIG. 5 by starch gelelectrophoresis according to the method of Harada et al., 1980, Am. J.Hum. Genet. 32:8-15, but were still heavily contaminated by non-ALDHproteins. The individual ALDH isozymes were pooled and dialyzed againstthe same buffer and were rechromatographed on an analytical DEAE 5PWHPLC column. For ALDH-I, elution was affected with the following NaClgradient: 0-160 minute, 0-500 mM, linear; 160-190 minute, 500 mM,isocratic; 190-195 minute, 500-0 mM, linear. The results of arepresentative elution profile in the isolation of ALDH-I is shown inFIG. 6. For ALDH-II, a shallower NaCl gradient was used: 0-80 minute,0-200 mM, linear; 80-100 minute, 200-500 mM, linear; 100-130 minute, 500mM, isocratic; 130-135 minute, 500-0 mM, linear. The results of arepresentative elution profile in the isolation of ALDH-II is shown inFIG. 7. The ALDH isozymes after second HPLC were about 95-98% pure asjudged by SDS gel electrophoresis (Laemmli and Favre, 1973, J. Mol.Biol. 80:575-599) and were used for kinetic analysis as described inExample 3.

EXAMPLE 3

Inhibition of ALDH by Daidzin and Related Compounds

Although daidzin is a known major constituent of Pueraria lobata(Nakamoto et al., 1977, supra; Chen and Zhang, 1985, supra) and otherplants (Eldridge and Kwolak, 1983, J. Agric. Food & Chem. 31:394-396),its effects on human alcohol metabolism were unknown. In particular, itsability to inhibit ALDH has not previously been reported or suggested.

The kinetic properties of daidzin toward human ALDH-I and ALDH-II werestudied using formaldehyde as substrate. The most commonly usedsubstrate, acetaldehyde, has an extremely low K_(m) value (˜2 μM) forALDH-I which does not permit accurate analysis of its kinetics by thespectrophotometric method. The K_(m) values of formaldehyde for ALDH-Iand ALDH-II, as determined in the present study, were 800 μM and 6.6 mM,respectively. These values are within the same range to those reportedfor the horse mitochondrial and cytosolic ALDH isozymes respectively(Pietruszko, 1989, supra).

The inhibition kinetics were studied by the initial velocity method(Dixon and Webb, 1979, Enzymes, 3rd ed., Longman, Great Britain).Daidzin was dissolved at different concentrations in methanol and wasadded to the assay medium as 10 μL aliquots. For controls, 10 μL ofmethanol was added to the assay medium. The initial reaction rates weremeasured in a pH 9.5 assay medium 0.1M in glycine-NaOH, 0.15M in KCl, 1mM in NAD⁺, 1% in methanol, 10 nM in ALDH-I or ALDH-II and variousconcentrations of formaldehyde and daidzin. The enzyme reaction rateswere followed by monitoring the production of NADH at 340 nm (ε=6.22mM⁻¹ cm⁻¹) with a Varian Cary 219 spectrophotometer thermostated at 25°C. The kinetic data were analyzed by standard graphical methods. Type ofinhibition and Michaelis constants for formaldehyde were analyzed byLineweaver-Burk plots and the inhibition constants were estimated byDixon plots (Dixon and Webb, 1979, supra). The kinetic parameters aresummarized in Table II.

                  TABLE II                                                        ______________________________________                                        Kinetic Constants for Daidzin Inhibition of                                   Human ALDH-I and ALDH-II Isozymes                                                    Isozyme K.sub.i (nM)                                                   ______________________________________                                               ALDH-I  40                                                                    ALDH-II 20,000                                                         ______________________________________                                    

Daidzin selectively inhibits ALDH-I at nanomolar concentrations. Thedata shows a 500-fold more effective inhibition of human ALDH-I than ofhuman ALDH-II by daidzin.

Inhibition of ALDH-I by daidzin is reversible. Preincubation of ALDH-Iwith 100 nM daidzin results in 70% inhibition which is reversed by100-fold dilution to yield a final inhibition of 2%. These facts pointto daidzin as a safe, effective and reversible means to achieve alcoholintolerance along the lines naturally available to ALDH-I deficientOriental individuals.

By the same token, genistin (Table III) is also a safe, effective andreversible selective inhibitor of ALDH-I, although nearly an order ofmagnitude less potent than daidzin.

                  TABLE III                                                       ______________________________________                                        Kinetic Constants for Prunetin and Genistin Inhibition                        of Human ALDH-I and ALDH-II Isozymes                                                       K.sub.i (nM)                                                     Isozyme        Prunetin Genistin                                              ______________________________________                                        ALDH-I         300      360                                                   ALDH-II        12,000   *                                                     ______________________________________                                         *No inhibition was observed up to 20 μM.                              

A survey of the inhibitory properties of commercially availablecompounds that are structurally similar to daidzin revealed only a fewthat inhibit ALDH-I as shown in Table IV, including an isoflavone, 3flavones, a chromone, a coumarin, a dihydrocoumarin and ahexahydrocoumarin, but none as potent as daidzin. None of these areknown components of RP as is daidzin. Moreover, none of these ALDHinhibitory compounds are potent yet highly selective inhibitors ofALDH-I as is daidzin, rather they show pronounced inhibition of ALDH-II.

                                      TABLE IV                                    __________________________________________________________________________    Structurally Related Compounds That Inhibit ALDH                                       Substituents                          IC.sub.50 (μM)              Type     2  3 4  5  7   8  4' Name             ALDH-I                                                                             ALDH-II                   __________________________________________________________________________    Isoflavone                                                                             H    ═O                                                                           H  OGlc                                                                              H  OH Daidzin          0.15 20                                 H    ═O                                                                           OH OMe H  OH Prunetin         1    12                                 H    ═O                                                                           OH OGlc                                                                              H  OH Genistin         2    #                                  H    ═O                                                                           H  H   H  i-Pr                                                                             4'-Isopropylisoflavone                                                                         5    1.5                                H    ═O                                                                           H  OGlc                                                                              H  OMe                                                                              Ononin           †                                                                           20                        Flavone     H ═O                                                                           H  H   H  H  Flavone          10   5                                     Ph                                                                              ═O                                                                           H  OH  H  H  3-Phenyl-7-hydroxyflavone                                                                      10   2                                     H ═O                                                                           OH OH  H  OMe                                                                              Acacetin         5    5                         Chromone Me Bz                                                                              ═O                                                                           H  OAc OAc   3-Benzyl-7,8-diacetoxy-2-                                                                      10   3                                                       methylchromone                                  Coumarin ═O                                                                           H Ph H  OH  H     7-Hydroxy-4-phenylcoumarin                                                                     10   0.3                       Dihydrocoumarin                                                                        ═O                                                                           H Ph H  Me  H     7-Methyl-4-phenyl-3,4-                                                                         10   0.5                                                     dihydrocoumarin                                 Hexahydro-                                                                             ═O                                                                           Ph                                                                              Me H  Cl  H     7-Chloro-4A,5,6,7,8,8A-hexahydro-                                                              1    1                         coumarin                      4-methyl-3-phenylcoumarin                       __________________________________________________________________________     *ALDH activities were assayed at 25° C. in 0.1M sodium                 pyrophosphate buffer pH 9.5 containins 0.15M KCl, 1 mM                        NAD.sup.+, various concentrations of inhibitors and 5 and 200 μM           acetaldehyde for ALDHI and ALDHII, respectively.                              #Does not inhibit ALDHII at up to 20 μM. †IC.sub.50 is greater      than 20 μM.                                                           

Most of the flavones and isoflavones tested for ALDH inhibitory activitydid not inhibit ALDH-I or ALDH-II as shown in Table V. Some of these areknown components of RP and other related compounds as indicated in TableV. In addition, allantoin, a component of RP, and 1-methylhydantoin, astructural analog of 5-methylhydantoin, which are not structurallyrelated to the compounds of Table V, are not inhibitory. Also2-phenylquinoline a steric analog of isoflavone did not inhibit.

For the study of the inhibition of ALDH-I by structurally relatedcompounds to generate the data shown in Tables IV and V, each individualcompound was dissolved at different concentrations in methanol and wasadded to the assay medium as 10 μL aliquots. For controls, 10 μL ofmethanol was added to the assay medium. In a standard ALDH-I assay, theassay medium contains 0.1M sodium pyrophosphate pH 9.5, 0.15M KCl, 1 mMNAD⁺, 1% methanol, 5 μM acetaldehyde, 5-10 nM ALDH-I and variousconcentrations of inhibitor. The enzyme reaction rates were measured bymonitoring the production of NADH at 340 nm (ε=6.22 mM⁻¹ cm⁻¹) with aVarian Cary 219 spectrophotometer thermostated at 25° C. (Fong et al.,1989, supra). The inhibition of ALDH-I by the inhibitors was calculatedas: ##EQU2## where V_(o) is the enzyme reaction rate measured in theabsence of inhibitor, and where V_(i) is the enzyme reaction ratemeasured in the presence of inhibitor. The inhibitor concentration thatproduces 50% inhibition is defined as IC₅₀, a parameter that is usefulfor comparison of inhibition of structurally related compounds as shownin Tables IV and V. The same procedure was used for measurement ofALDH-II as for ALDH-I, except that 200 μM acetaldehyde was used in theassay medium instead of 5 μM. IC₅₀ values are related to the underlyingK_(i) values. For competitive inhibitors at constant initialconcentration or substrate, [S₀ ], for example, daidzin, prunetin,genistin, and likely their analogs, the relevant formula is:

    IC.sub.50 =((3[S.sub.0 ]/K.sub.m)-1)K.sub.i.

In Table IV, [S₀ ] is approximately equal to 2.5 times K_(m) for ALDH-Iand therefore IC₅₀ equals 6.5 times K_(i) ; similarly [S₀ ] equals K_(m)for ALDH-II measurements and IC₅₀ equals 2 times K_(i).

                                      TABLE V                                     __________________________________________________________________________    Structurally Related Compounds That Do Not Inhibit ALDH†               Substituents                                                                  Type  2  3     4  5   6  7   8   2' 3'  4' 5'  Name                           __________________________________________________________________________    Isoflavone                                                                          H        ═O                                                                           H   H  OH  H   H  H   OH H   Daidzein*                            H        ═O                                                                           H   H  OH  H   H  H   OMe                                                                              H   Formononetin*                        H        ═O                                                                           OH  H  OH  H   H  H   OH H   Genistein*                           H        ═O                                                                           OH  H  OH  H   H  H   OMe                                                                              H   Biochanin A*                         H        ═O                                                                           H   H  OH  OGlc                                                                              H  H   OH H   Puerarin*                            Me       ═O                                                                           H   H  OAc H   H  H   H  H   7-Acetoxy-2-methylisoflavon                                                   e                                    Me       ═O                                                                           H   H  OAc OAc H  H   H  H   7,8-Diacetoxy-2-methylisofl                                                   avone                          Isoflavan                                                                           H        H  H   H  OH  H   H  H   OH H   Equol                          Flavone  H     ═O                                                                           H   H  H   H   Cl H   H  H   2'-Chloroflavone                        H     ═O                                                                           H   H  OH  H   H  H   H  H   7-Hydroxyflavone                        H     ═O                                                                           H   H  O.sub.2 CPh                                                                       H   H  H   H  H   7-Benzoyloxyflavone                     H     ═O                                                                           H   H  OH  OH  H  H   H  H   7,8-Dihydroxyflavone                    H     ═O                                                                           OH  H  OH  H   H  H   H  H   Chrysin                                 H     ═O                                                                           OH  H  OMe H   H  H   H  H   Techtochrysin                           H     ═O                                                                           OH  H  OH  H   H  H   OH H   Apigenin                                OH    ═O                                                                           H   H  H   H   H  H   H  H   3-Hydroxyflavone                        OH    ═O                                                                           OH  H  OH  H   H  H   H  H   Galangin                                OH    ═O                                                                           OH  H  OH  H   H  H   OH H   Kaempferol                              OH    ═O                                                                           H   H  OH  H   H  OH  OH H   Fisetin                                 OH    ═O                                                                           OH  H  OH  H   OH H   OH H   Morin                                   OH    ═O                                                                           OH  H  OH  H   H  OH  OH H   Quercitin                               O-rutinose                                                                          ═O                                                                           OH  H  OH  H   H  OH  OH H   Rutin                                   OH    ═O                                                                           OH  H  OH  H   H  OH  OH OH  Myricetin                      Flavan   H     ═O                                                                           H   H  H   H   H  H   H  H   Flavanone                               H     ═O                                                                           OH  H  OH  H   H  H   OH H   4',5,7-Trihydroxyflavanone                                                    .                                       OH    H  OH  H  OH  H   H  OH  OH H   (+/-)-Catechin                          OH    H  OH  H  OH  H   H  OH  OH H   (-)-Epicatechin                Coumarin                                                                            ═O                                                                           #     OH H   H  H   H                 Warfarin                             ═O                                                                           H     H  H   OMe                                                                              OMe H                 6,7-Dimethoxycoumarin*         __________________________________________________________________________     *Present in Radix Puerariae. #2Acetyl-1-phenylethyl. †No inhibitio     observed at 20 μM inhibitor.                                          

EXAMPLE 4

In Vivo Effects of Daidzin on Alcohol Consumption

In order to demonstrate the in vivo effect of daidzin on alcoholconsumption, experiments were designed to test the effect of RP extracton free choice ethanol intake in golden hamsters. Hamsters were chosenbased on previous reports that they are receptive to and give preferenceto high ethanol intake when compared with several other mammalianspecies. Hamsters drink significant quantities of ethanol inconcentrations up to 70% (w/v) under free choice conditions, and drinkvirtually all of their daily water as ethanol at concentrations below15%. Daily dosages of ethanol during free-choice consumption by hamstersranges from 10 to 16 g/kg/day, equivalent to a daily consumption of 6.5ml to 10.4 ml of 20% ethanol for a 130 g hamster (Kulkosky and Cornell,1979, Pharmacol. Biochem. & Behav. 11:439-44). There is an inversecorrelation of ethanol concentration with volume of ethanol solutionconsumed (Kulkosky and Cornell, 1979, supra) such that 20-30% ethanol isan appropriate concentration range over which to obtain accurate volumereadings in free choice experiments.

The animals used for the experiments described in this example were sixmale adult golden hamsters (outbred, Lakeview Lak: LVG[SYR]), purchasedfrom Charles River Laboratories, Wilmington, Mass. 01887. Animals weremaintained on a 12/12 light/dark cycle (light on 0600-1800 hr) for aperiod of 6 weeks. Animals had access to food and water ad libitum.

An RP extract was prepared as follows: Dried RP, 100 g, was ground to apowder in a domestic food processor and was refluxed with 1 liter ofmethanol overnight in a 2 liter round bottom flask equipped with areflux condenser, as described in Example 1. The methanol extract waspassed through Whatman No. 1 filter paper to remove debris, methanol wasremoved from the extract by vacuum evaporation and the resultant syrup(˜16 g) was suspended in 16 ml of water.

For the experiment, 6 animals were maintained as described above on asingle large cage with four 250 ml calibrated drinking bottles. Thedrinking bottles were fitted with stainless steel straight sipper tubes,used to measure fluid consumption to the nearest 5 ml. Spillage from thedrinking tubes was caught by 2 oz. jars fitted with glass funnels andpositioned under the sipper tubes. Fluid consumption by the 6 hamsterswas measured once every 3 days so that the consumption volumes werelarge enough to obtain reasonably accurate measurements.

After a 6-week acclimation period, the body weights of the animalsreached ˜180 g and stayed generally unchanged throughout the experiment.Total water intake of the animals was also stabilized at about 12ml/day/animal as shown in FIG. 8a. Water in 2 of the 4 drinking bottleswas then replaced by a 15% ethanol solution and consumption of water andaqueous ethanol were measured for a period of 2 weeks. Within 2 to 3days after the beginning of this free choice phase of feeding, thehamsters had established an explicit preference for aqueous ethanol overwater with a preference ratio (defined as aqueous ethanol intake dividedby water intake) of about 8 to 9, which stayed fairly constantthroughout the next 2 weeks.

As a control, the animals were then fed with 0.2 ml water twice daily,using a stainless steel animal feeding needle. Water feeding did notseem to have any effect on the animals' drinking behavior as measured bytotal fluid intake (FIG. 8a). After 6 days, the same group of hamsterswere fed with 0.2 ml of RP extract (containing 1.6 mg of daidzin asanalyzed by HPLC) twice daily. The extract had a dramatic effect on thepreference ratio of the hamsters. As shown in FIG. 8b, except for day9-12, the preference ratios were substantially lower when the animalswere on the RP extract regimen. At day 39, feeding of RP extract wasterminated and the preference ratio returned to normal. While RP had adramatic effect on preference ratio (FIG. 8b), the total fluid intakewas not affected (FIG. 8a).

EXAMPLE 5

In Vivo Effects of Daidzin on Alcohol Consumption

In order to demonstrate that daidzin decreases alcohol consumption invivo, experiments were designed to test the effect of daidzin on freechoice ethanol intake in golden hamsters. Hamsters were chosen asdescribed in Example 4 based on previous reports that they display highethanol intake and preference in comparison with several other mammalianspecies, and that this was correlated with differences in ethanolmetabolism. Kulkosky and Cornell, 1979, supra.

The animals used in these experiments are adult male golden hamsters(outbred, Lakeview Lak: LVG[SYR]), purchased from Charles RiverLaboratories, Wilmington, Mass. 01887. Upon arrival, hamsters are housed(4 per cage) with ad libitum access to food and tap water in a roommaintained at 23° C. on a 12/12 light/dark cycle (light on 0600-1800hr.) for 1 week. Following this acclimation period, each hamster istransferred to an individual stainless steel metabolic cage (26×18×17.5cm) with a wire mesh floor for the remainder of the experiment. Eachcage is equipped with a stainless steel food hopper located on the rightside of the front wall which was kept filled with food. Two 50 mldrinking bottles fitted with stainless steel sipper tubes are placed onthe left side of the front wall. Under the sipper tubes are funnelswhich collect and direct spillage to tubes placed outside of the cages.Fluids are provided mainly during the dark cycle (1800-0800 hr.) andfluid intake is measured in the morning at the same time each day, forexample, at 0800 hr. During this baseline period, for example, 8 days,two drinking solutions are provided for each animal. One is tap water;the other is a 30% v/v solution of ethanol (100% USP). The position ofthe two drinking bottles on each cage is alternated daily to prevent thedevelopment of positional preference. Hamsters that drink significantamounts of ethanol solution, for example more than 5 ml/day, and thatdisplay consistent water and ethanol consumption are selected fordaidzin administration after the pretreatment period.

In one experiment, two of eleven hamsters tested during a pretreatmentperiod of 8 days drank more than 5 ml of ethanol solution per day anddisplayed the most consistent water and ethanol consumption. These twowere selected on the last day (day 8) of the pretreatment period for thestudy of the effect of daidzin. One of them (Number 6) exhibited astrong preference for ethanol solution (ethanol vs. water intake ratio7.6); the other hamster (Number 9) displayed virtually no preferencebetween water and ethanol solution (ethanol vs. water intake ratio 1.1)(FIGS. 9a,b).

In this experiment, at 0900 hr. of day 9, the two hamsters selected(Numbers 6 and 9) received a single dose of 10 mg daidzin (as a 0.5 mLsuspension in saline, subcutaneously) and in the following 13 days, eachhamster was fed daily at 1700 hr. 10 mg of daidzin suspended on 0.5 mLwater, using a stainless steel animal feeding needle. In otherexperiments, subcutaneous administration is preferably omitted, and eachhamster receives only daily oral administration by feeding as describedherein. As shown in FIG. 9a, alcohol intake by hamster Number 6 startedto decline 2 days after the first dose of daidzin. This decline inalcohol intake was accompanied by a concomitant increase in waterintake. While the total fluid intake was slightly decreased during theperiod of daidzin administration, more importantly, the total H₂ Ointake, that is, the sum of water from the water bottle and water in theethanol mixture, stayed nearly constant. A similar result was observedin hamster Number 9 except that in this animal, the effect of daidzindid not become apparent until the fourth day after the first dose wasgiven as shown in FIG. 9b. Feeding of daidzin was terminated at 1700 hr.on day 20. The nearly constant and low ethanol preference ratios thatcharacterized the last 70 to 75% of the treatment phase and the firsttwo days of the posttreatment phase (Number 6, 0.40±0.04 SEM, days 14 to22; Number 9, 0.31±0.02 SEM, days 13 to 22) increase significantly forthe balance of the posttreatment phase approximately to the level of nopreference for ethanol or water (Number 6, 1.19±0.11 SEM, days 23 to 38;Number 9 0.89±0.05 SEM, days 23 to 38).

In our experience, golden hamsters obtained from various U.S. sourcesundergo a cyclic change in alcohol preference Specifically, in ourexperience, eighty to ninety percent of hamsters received from Maythrough September did not prefer ethanol solutions, whereas at othertimes of the year nearly all hamsters showed a strong preference forethanol. We are not aware of previous reports of this behavior in theliterature. All hamsters in this and following examples of drinkingbehavior, except for Example 9, were preselected for alcohol preference,as described in Example 4.

During May to September, in those cases where no hamsters were found toprefer ethanol by free choice as described in Example 4, it wasgenerally possible to train some of them to prefer ethanol by providinga 20% ethanol solution and no water for 10 to 20 days, after which theyshowed their trained preference for ethanol under free choiceconditions. Such animals were used for the experiments described inExample 9. This methodology is commonly used to train outbred rats toconsume ethanol (see, e.g., Samson, et al., 1988, Alcoholism: Clin. Exp.Res. 12:591-598).

EXAMPLE 6

Dose Effects on Alcohol Consumption of Daidzin Administered as a PureCompound and as a Component of Crude RP Extract

It is common practice in quantitative drug studies with rodents to useintraperitoneal (i.p.) administration to deliver drug doses accurately(Goodman and Gilman, 1975, The Pharmacological Basis of Therapeutics,5th ed., Macmillan, N.Y., p. 8). In particular, this method avoidsvariability introduced by technical difficulties of oral administrationvia feeding (full versus empty stomach, etc.), variations in drugtransport in the gastrointestinal tract (variations in intestinal faunaand flora, individual variations in absorption rate expected of outbredanimals, etc.) and other factors. Hence, a quantitative dose effectexperiment was performed as in Example 5 but with daidzin as the druginjected i.p. as a suspension in 1 ml of sterile saline. As a control 1ml of sterile saline was injected i.p. daily during the baseline period(typically 7-9 days) that establishes drinking preference without thedrug. Saline injection i.p. had no effect on the drinking behavior ofgolden hamsters. Ethanol was provided as a 20% solution. Hamsters couldbe used more than once (but were not used more than three times) in acycle of baseline saline injections followed by drug injections, eachcycle with the drug at a different dose.

The response to daidzin treatment was computed as:

    % Response=(V.sub.o -V.sub.d)×100 / V.sub.o

where V_(o) is average daily intake of 20% ethanol during the baselineperiod and V_(d) is the average daily intake during the daidzintreatment period.

The right-hand curve of FIG. 10 (solid circles) shows a graded-doseresponse of pure daidzin on golden hamster alcohol intake. Data in thesemilog plot are shown as mean±SE for 6 to 9 different determinations.The slope is typical of dose response curves. Daidzin at a dose of 5mg/day suppresses hamster alcohol intake by 20%; suppression increasesto 50% (i.e., EC₅₀ or half-maximal response) at 10 mg/day and increasesfurther to 80% at 30 mg/day.

When this experiment was repeated with crude RP methanolic extractinstead of pure daidzin the dose-response curve for daidzin in the crudeextract was shifted left from the curve for pure daidzin as shown in theleft-hand curve of FIG. 10 (solid squares) to an EC₅₀ value 5 to 10times lower than that for pure daidzin. Hence, daidzin in the crudeextract is 5 to 10 times more potent than pure daidzin. Among thepossible explanations for this effect are any combination of thefollowing: crude RP extract may contain additional active principle(s)that may act either additively or synergistically with daidzin insuppressing alcohol intake; or additional principle(s) in the crude RPextract may increase the bioavailability of daidzin and may act forexample, by promoting its absorption intraperitoneally and/or itstransport across biomembranes generally. As demonstrated in thefollowing example, the RP extract contains a factor or factors whichincrease the bioavailability of daidzin as determined by theadministration of daidzin in the extract as compared with administrationof purified daidzin.

EXAMPLE 7

Relative Bioavailability of Daidzin: Pure Daidzin vs Daidzin in Crude RPExtract

In hamsters, daidzin administered i.p. as a crude RP extract has ahigher relative bioavailability than pure daidzin as demonstrated in thefollowing experiment.

Nine hamsters (130±10 g) were partially anesthetized with 1 mL of 30%ethanol i.p., and 30 min later, four of these were injected i.p. withdifferent doses (2, 4, 8 or 30 mg) of pure daidzin suspended in 1 mLsterilized saline. The remaining five received different doses (30, 100,200, 300 or 400 mg) of crude extract containing 0.24, 0.8, 1.6, 2.4 or3.2 mg of daidzin in the same fashion. Blood samples, taken at intervalsfrom the orbital venosus plexus, were collected in heparinized tubes.Plasma was obtained by centrifugation at 90000 rpm in a Beckman Airfuge.Plasma proteins were precipitated with an equal volume of acetonitrileand removed by centrifugation. The daidzin content of the supernatantsolution was analyzed by HPLC. Plasma daidzin concentration-time curveswere constructed from these data and the area under curve (AUC) inμM.min was estimated and defined as relative bioavailability. Therelative bioavailability as shown in FIG. 11 of daidzin given in theform of crude extract (solid circles) is about 9 times that of daidzinadministered as a pure compound (solid squares). When the dose-responsecurves of FIG. 10 were replotted using relative bioavailability ratherthan the daidzin doses given, the two curves virtually merged as shownby FIG. 12. In FIG. 12, the curve for daidzin administered as crudeextract is represented by solid squares, whereas the curve for purifieddaidzin is represented by solid circles. These results not only indicatethat daidzin in the crude RP extract is more readily available to theanimal system, but also lends support to the proposal that daidzin isthe major, if not the only, active principle in RP that suppressesalcohol intake. The increased bioavailability of daidzin given as thecrude RP extract appears to be due to a factor or factors in theextract. Such a factor or factors may act by promoting solubility ofdaidzin in aqueous solution. For example, a saturated water solution ofpure daidzin is 0.2 mM whereas a saturated water solution of previouslydried RP methanolic extract, or of previously dried RP 95% ethanolicextract prepared as for the RP methanolic extract, is 7 mM in daidzin.

The following pharmaceutical solubilizing agents, obtained from SigmaChemical Co., St. Louis, Mo., also increased the solubility of daidzinin water. Daidzin was highly soluble both in glycerol and inpolyethylene glycol 400 (PEG). When a concentrated solution of daidzinin PEG was diluted with water to 20% PEG/H₂ O, daidzin solubility was4.5 mM. When β-cyclodextrin is dissolved in the above solution ofdaidzin in PEG at a concentration that yields 20 mg/ml of β-cyclodextrinafter dilution to 20% PEG/H₂ O, daidzin solubility was 9.4 mM. Thesolubility of daidzin in 10% aqueous polyvinylpyrrolidone 40 and in asaturated solution of a representative saponin, digitonin, was about 2mM.

Different strategies may be employed to increase the bioavailability ofdrugs, of which several examples are given. These include makingderivatives (i.e., analogs) of higher water solubility as in Example 8,using agents known to increase water solubility of drugs, e.g.,polyethylene glycol, cyclodextrin, polyvinylpyrrolidone, saponins, etc.,and/or isolating new agents, particularly from the RP extract, thatincrease water solubility of daidzin specifically or isoflavones orflavones in general.

EXAMPLE 8

Inhibition of ALDH by Novel Compounds Related to Daidzin

Novel compounds derived from daidzein (i.e., daidzin analogs) byreaction with various ω-bromo fatty acids or with ethyl iodide have beenfound unexpectedly to mimic the inhibitory properties of daidzin. Thesenovel compounds are named as ethers of the 7-hydroxyl group of theaglycone daidzein; they resemble daidzin in that there is no freehydroxyl at the 7-position, but differ in that the daidzin glucosidicgroup is an acetal rather than an ether.

Daidzein (10 mmoles) was suspended in 40 ml of acetone, 10 ml of 2N KOHwas added, followed by 10 mmoles of solid ω-bromohexanoic acid,ω-bromoheptanoic acid, ω-bromoundecanoic acid or ethyl iodide. Themixture was stirred under reflux for 3 days.

The potassium salt of the 7-(ω-carboxyalkyl) ether of daidzein wasrecovered by filtration, washed with acetone and dried; yield, 20-35%.The resultant white crystals were more than 95% pure and contained onlyabout 1% daidzein as a contaminant, as determined by HPLC on a Watersradial pack C18, 5 μm, 0.8×10 cm column with a linear 15-min gradientelution from 0.1% trifluoracetic acid to 80% acetonitrile/0.1%trifluroacetic acid at 1 mL/min monitored at 254 nm. Unreacted daidzeinwas the principal isoflavone component of the filtrate and can berecovered by acidification, filtration and recrystallization fromethanol. Retention times in the above HPLC system were: daidzin 13.92,daidzein 15.33, daidzein 7-(ω-carboxypentyl) ether 17.67, daidzein7-(ω-carboxyhexyl) ether 18.27 and daidzein 7-(ω-carboxydecyl) ether20.37 min. The longer retention times of the novel compounds areconsistent with their lower polarity. Mass spectral analysis, performedas in Example 1, revealed molecular ion peaks at m/z 368 for thecarboxypentyl derivative and at m/z 382 for the carboxyhexyl derivative.Daidzein peaks from decomposition of the molecular ions, at m/z 118,137, 253 and 254 (Ganguly and Sarre, 1970, supra) were also evident.Ultraviolet absorption spectra of daidzein 7-(ω-carboxyhexyl) ether atpH 7.4 showed maxima at 250 and 304 nm, and no maximum at 331 nm that ischaracteristic of a free 7-hydroxyl group in daidzein. The spectrum atpH 11 showed maxima at 245, 250 and 280 nm characteristic of alkylationof the 7-OH group of daidzein as occurs in daidzin; daidzein at this pHhas easily distinguishable maxima at 331 and 260 nm. The monopotassiumsalts of the carboxyalkyl derivatives were more soluble in water thanwas daidzin (0.2 mM): daidzein 7-(ω-carboxypentyl) ether 26 mM, daidzein7-(ω-carboxyhexyl) ether 13 mM, daidzein 7-(ω-carboxydecyl) ether 9 mM.The free acids precipitated when the pH was adjusted to 2 with 1N HCl.Methanol was added to each of the suspensions, which were heated untilthe precipitates dissolved. Upon cooling white crystals of daidzein7-(ω-carboxypentyl) ether mp 223-225 and daidzein 7-(ω-carboxyhexyl)ether mp 193-198 formed, whereas daidzein 7-(ω-carboxydecyl) etherseparated as an oil.

The H¹ NMR spectra of the carboxypentyl and carboxyhexyl compounds in d₆-DMSO exhibited the high field resonances above 6 ppm expected fordaidzin but not daidzein, as shown in Example 3. In the low field regionthere were multiplet resonances between 1.1 and 2.3 ppm thatcorresponded to 4 methylene groups in the carboxypentyl derivative and 5in the carboxyhexyl derivative and a triplet at 4.1 ppm corresponding totwo methylene protons adjacent to the ether linkage.

Daidzein 7-ethyl ether was obtained as a byproduct of the synthesis ofdaidzein by condensation of the hydroxyphenyl hydroxybenzyl ketone withethyl orthoformate (Iyer et al., 1951, Proc. Ind. Acad. Sci. 33A:116).This byproduct has not been previously recognized or reported. Ananalogous byproduct has been reported in the synthesis of7-ethoxyisoflavone (Mester et al., 1991, Chem. Abstract 115:158826b,Hung. Teljes HU 55,376 Abstract). The identity of the novel byproduct ofthe above ethy iodide reaction was established by HPLC; the retentiontime was 17.83 min. The byproduct exhibited the expected molecular ionpeak at m/z 282 and daidzein-related peaks at 118, 137, 253 and 254.Ultraviolet spectra at pH 7.4 (maxima 250 and 304 nm) and pH 11 (245,250 and 280 nm) were consistent with alkylation of the 7-OH position andnot the 4'-OH position of daidzein.

The progress curve method described by Klyosov and Berezin, 1972,Biokhimiya (Engl. transl.), Plenum, New York, 37:141-151, was usedinstead of the initial velocity method of Example 3 to obtain precisevalues for inhibition constants for ALDH-I with acetaldehyde assubstrate and the very tight binding novel inhibitors (Table VI).

                  TABLE VI                                                        ______________________________________                                        Competitive Inhibition of ALDH Isozymes by Daidzin                            and Analogs with Acetaldehyde as Substrate                                                      K.sub.i, nM                                                                   ALDH-I  ALDH-II                                             ______________________________________                                        Daidzin             42        28000                                           Daidzein 7-ethyl ether                                                                            38        440                                             Daidzein 7-(ω-carboxypentyl) ether                                                          9         450                                             Daidzein 7-(ω-carboxyhexyl) ether                                                           9         215                                             Daidzein 7-(ω-carboxydecyl) ether                                                           3         185                                             ______________________________________                                    

All of the novel compounds are as potent as, or more potent than daidzinas inhibitors of ALDH-I, and all are selective for ALDH-I over ALDH-II,but none is as selective as daidzin: the ratio K_(i), ALDH-II /K.sub.,ALDH-1 is 667 for daidzin, but is 24-62 for the carboxyalkylderivatives.

EXAMPLE 9

In Vivo Effects of Daidzein 7-(ω-Carboxyhexyl) Ether and Daidzein7-(ω-Carboxypentyl) Ether on Alcohol Consumption

Daidzein 7-(ω-carboxyhexyl) ether and daidzein 7-(ω-carboxypentyl) etheralso suppress alcohol intake in vivo. The experimental procedure was thesame as for Example 6 except that trained hamsters described in Example5 were used and the carboxyalkyl ether derivatives were tested at onlyone dose, 10 mg/day, which is the EC₅₀ for daidzin. At that dosedaidzein 7-(ω-carboxyhexyl) ether suppresses golden hamsters alcoholintake by 72% and 69% in two hamsters (E23 and B7, respectively), anddaidzein 7-(ω-carboxypentyl) ether suppresses intake by 72% and 56% intwo hamsters (D6 and C14, respectively). Assuming a dose response slopesimilar to that of daidzin, both carboxyalkyl derivatives are not morethan twice as potent as daidzin, which is within the range ofexperimental error.

EXAMPLE 10

Relative Bioavailability: Daidzin vs. Daidzein 7-(ω-Carboxyhexyl) Ether

The drug-time curves as shown in FIG. 13 for daidzein 7-(ω-carboxyhexyl)ether (solid squares) and daidzin (solid circles) in plasma, determinedaccording to the same procedure as in Example 7 but at doses of 10 mgfor each hamster, are also very similar to each other. Both compoundsreached their maximal plasma concentrations (4.5 and 6 μM for thecarboxyhexyl ether and daidzin, respectively) within about an hour afterinjection. Five hours later the plasma concentrations of both inhibitorswere still above 2 μM, far above their competitive inhibition constantsfor ALDH-I.

What is claimed is:
 1. A method for therapeutically treating alcoholconsumption in a human comprising administering a pharmaceuticalcomposition comprising daidzin in an effective amount to reduce alcoholconsumption and a pharmaceutical carrier.
 2. A method according to claim1 wherein the composition induces a sensitivity reaction after alcoholconsumption.
 3. A method according to claim 1 wherein the human is analcoholic.
 4. The method of claim 1 wherein the effective amount isbetween about 0.1 mg to about 140 mg per kilogram of body weight.
 5. Amethod for therapeutically treating alcohol consumption in a humancomprising administering an amount of daidzin effective to reducealcohol consumption.
 6. The method of claim 5 wherein the amount ofdaidzin effective to reduce alcohol consumption is between about 0.1 mgto about 140 mg per kilogram of body weight.